This study is based on the anti-lipopolysaccharide factor-like (ALF-like) gene (Anti-lipopolysaccharide factor-like) identified by our research group from transcriptomic databases of different disease-resistant materials (highly resistant and lowly resistant). The anti-lipopolysaccharide factor plays a significant role in immune defense, inflammatory regulation, and lipid metabolism. In-depth investigation of its mechanism of action could provide important theoretical support for the development of therapeutic strategies for related diseases. Therefore, this study explores the functional role of the ALF-like gene in Penaeus vannamei in resisting white spot syndrome virus (WSSV). In our previous WSSV infection experiments, it was observed that the expression of this gene is upregulated in hemocytes, hepatopancreas, gills, and intestine tissues. Based on this finding, this study employs RNA interference (RNAi) technology to knock down the ALF-like gene. Based on this, in this study, RNAi technology was used to knock down the ALF-like gene. The expression levels of apoptosis and antioxidant pathways in intestinal tissues were detected by real-time fluorescence quantitative PCR technology, and the proliferation change rules of WSSV in the shrimp body were analyzed. Additionally, PCR (Polymerase Chain Reaction, PCR) and Sanger sequencing techniques were used to analyze the changes in single nucleotide polymorphism (SNP) sites between the amplified sequences and the original NCBI sequences. Furthermore, an association analysis of WSSV resistance and SNP sites, as well as KASP (Kompetitive Allele-Specific PCR) typing verification, were conducted. This study provides a theoretical basis for analyzing the molecular mechanism of white spot syndrome and the molecular breeding of disease resistance in P. vannamei , which has certain reference value.