Litopenaeus vannamei is an economically significant species in aquaculture. As the scale of aquaculture continues to expand, high-density farming often leads to disease outbreaks and feed-related issues, which negatively impact shrimp health, product quality, and farming profitability. Astaxanthin, a powerful carotenoid antioxidant, is widely used in aquaculture to enhance body condition, pigmentation, and growth performance. Currently, astaxanthin is mainly derived from chemical synthesis or biological extraction, with differences in cost, structure, and safety. Synthetic astaxanthin is low-cost and commercially produced, but may contain non-natural by-products, requiring strict dosage control. Natural astaxanthin is primarily extracted from Haematococcus pluvialis or Phaffia rhodozyma. Meanwhile, for different aquatic products, before using astaxanthin as a feed additive, the source of astaxanthin and its optimal addition level should first be confirmed to reduce costs and improve aquaculture and economic benefits. The purpose of this study was to comprehensively evaluate and compare the effects of synthetic astaxanthin (Group A) and broken-wall Phaffia rhodozyma-derived astaxanthin (Group B, a natural source) on the growth performance, antioxidant capacity, immune response, intestinal health, and gene expression of L. vannamei through multi-index detection and multi-omics analysis. Specifically, the objectives included: (1) Determining the differences in growth promotion, antioxidant, and immune-enhancing effects of astaxanthin from different sources and concentrations by measuring growth indicators, antioxidant enzyme activities, and related gene expression levels, and selecting the optimal effect groups for subsequent experiments; (2) Clarifying the differences in the effects of astaxanthin from different sources on intestinal morphology and microbial community structure of L. vannamei in the optimal effect groups through comprehensive analysis of intestinal tissue morphology and intestinal microbiota; (3) Revealing the differences in the effects of astaxanthin from different sources on the hepatopancreas of L. vannamei at the gene level by performing transcriptome sequencing on the hepatopancreas of the optimal effect groups. Ultimately, this study aimed to identify the most suitable type and concentration of astaxanthin for the growth and development of L. vannamei, and to provide theoretical basis and data support for the precise nutrition strategy and efficient environmental protection feed development in shrimp aquaculture. In this study, healthy L. vannamei with an initial average body weight of (15.64±1.99) g and an average body length of (13.90±0.58) cm were selected as experimental subjects. After a 3-day acclimation period, the shrimp were randomly divided into 3 treatment groups with 3 replicate tanks each (30 shrimp per tank). The control group (Con) was fed a basal diet, while the experimental groups were fed basal diets supplemented with synthetic astaxanthin (A60, A90, A120 at concentrations of 60, 90, 120 mg/kg, respectively) or broken-wall P. rhodozyma-derived astaxanthin (B60, B90, B120 at concentrations of 60, 90, 120 mg/kg, respectively). The experimental feeding period lasted for 21 days. At the end of the feeding experiment, samples were collected after 24 hours of fasting. Growth indicators such as final body weight (FBW), weight gain rate (BWG), specific growth rate (SGR), feed utilization ratio (FUR), survival rate (SR), and protein efficiency ratio (PER) were measured. Hepatopancreas and intestinal tissues were dissected under sterile conditions: hepatopancreas samples were used for the determination of antioxidant enzyme activities (including total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), alkaline phosphatase (AKP), and total nitric oxide synthase (TNOS)) and gene expression analysis (using quantitative real-time PCR to detect the expression of immune-related genes such as proPO, LZM, Toll-1, Toll-2, and antioxidant-related genes such as SOD and CAT); intestinal tissues were fixed with paraformaldehyde for morphological observation (H&E staining) or stored at -80℃ for intestinal microbiota analysis (16S rRNA gene sequencing of the V3-V4 region) and functional prediction (using PICRUSt2). Based on the results of growth indicators, enzyme activities, and gene expression, the optimal effect groups (A120 and B120) were selected for hepatopancreas transcriptome sequencing (RNA-seq) to analyze differentially expressed genes (DEGs), and functional annotation and pathway enrichment analysis were performed using GO and KEGG databases. The reliability of transcriptome sequencing results was verified by quantitative real-time PCR. All experimental data were statistically analyzed using SPSS 26.0 and GraphPad Prism 10 software, with one-way ANOVA and Tukey's test for multiple comparisons, and P<0.05 considered statistically significant. The results of this study showed that both synthetic and broken-wall P. rhodozyma-derived astaxanthin had significant positive effects on L. vannamei, but there were obvious differences in their efficacy and mechanisms: (1) Astaxanthin supplementation significantly improved final body weight, weight gain rate, feed utilization, and protein efficiency, and increased survival rate at appropriate concentrations. Synthetic astaxanthin showed the best growth-promoting effect at 120 mg/kg, while yeast-derived astaxanthin significantly enhanced final body weight at 90 mg/kg, indicating higher bioactivity and utilization efficiency. (2) Both types of astaxanthin significantly enhanced total antioxidant capacity (T-AOC), particularly at 120 mg/kg. At this dose, the yeast-derived astaxanthin also significantly increased catalase (CAT) and acid phosphatase (ACP) activities, demonstrating more comprehensive antioxidant and immune-enhancing potential. (3) At the genetic level, astaxanthin significantly regulated the expression of immune-related genes such as prophenoloxidase (proPO) and antioxidant genes such as superoxide dismutase (SOD) and CAT. Yeast-derived astaxanthin, even at lower doses, caused significant downregulation of key genes in the Toll pathway, suggesting a unique immunomodulatory mechanism. (4) Both astaxanthin types improved intestinal morphology, but in distinct ways: the synthetic group significantly increased villus height but reduced basal layer thickness, while the yeast-derived group significantly increased basal layer thickness without markedly affecting villus height, indicating better structural integrity. Microbiota analysis showed that astaxanthin optimized microbial composition by increasing beneficial bacteria (e.g., tentative Bacillaceae) and reducing potential pathogens such as Vibrio. Yeast-derived astaxanthin showed stronger enrichment effects for certain beneficial bacteria. KEGG functional prediction indicated that synthetic astaxanthin had no significant impact on major microbial functions, whereas the yeast-derived group led to significant downregulation in most functional abundances except purine metabolism, ribosomes, and amino acid biosynthesis. (5) Transcriptome analysis revealed mechanistic differences: the synthetic group was enriched in metabolic pathways such as caffeine metabolism and fatty acid degradation, while the yeast-derived group was enriched in DNA replication, folate biosynthesis, and other cell proliferation and repair pathways. Notably, the enrichment of the "chemical carcinogenesis-DNA adducts" pathway in the synthetic group suggests potential biosafety concerns. GO analysis showed that the synthetic group was enriched in "chitin binding" and "transport activity," while the yeast-derived group was enriched in the "extracellular region" and "growth factor activity," consistent with phenotypic results. These differences may be attributed to the structural composition of the two astaxanthin types (synthetic astaxanthin is a 1:2:1 mixture of left-, meso-, and right-handed isomers, while yeast-derived astaxanthin is purely right-handed) and other components (e.g., non-natural by-products in synthetic astaxanthin; glucans and proteins in yeast-derived astaxanthin). This study provides a theoretical basis and data support for the rational application of synthetic and disrupted P. rhodozyma -derived astaxanthin in the healthy farming of L. vannamei, contributing to the sustainable development of shrimp feed industry.