Temperature is an important abiotic factor affecting the survival, growth and development of fish. Water temperature exceeding the adaptation temperature of fish will cause heat stress, which will trigger a series of physiological reactions and affect the normal function of various organs and systems. Heat stress has become an important factor restricting the development of sturgeon farming industry. How to effectively alleviate the adverse effects of heat stress on Huso dauricus has become an urgent problem to be solved in the field of sturgeon farming. Arginine is an essential amino acid for aquatic animals. It functions in the body in the form of physiologically active L-arginine. It has been proven to significantly improve the growth performance, product quality, antioxidant capacity and immune response of aquatic animals. However, the effect and mechanism of exogenous L-arginine on heat stress relief of Huso dauricus are still unclear. Therefore, we designed this experiment. A total of 270 Huso dauricus (5 months old) with good health and no obvious trauma were randomly divided into 9 indoor temperature controlled circulating aquiculture tanks. The average body weight and length of the huso were (23.42 ± 2.26) g and (14.45 ± 2.62) cm, respectively. During this period, the light-dark cycle was set to 12 h: 12 h, water temperature was (19.0 ± 0.5) ℃, pH was ~6.9, and dissolved oxygen was ≥ 6 mg/L. The daily feeding was carried out twice in the morning and evening at 7:00 and 19:00, and the daily feeding amount was 3% of the body weight (Shandong Shengsuo commercial pellet feed: crude protein ≥ 40, crude fat ≥ 10%, crude fiber ≤ 6%, crude ash ≤ 18%, moisture ≤ 12%). Water change were carried out before feeding in the morning, and the daily water change was 50%. After one week of acclimation, the experiment was set up with 19℃ control group, 28℃ heat stress group and 28℃ arginine (R) addition group. Each group was set up three parallel, and the culture density was 30 tails/tank. The water temperature was adjusted to the experimental temperature at 1℃/h after the start of the experiment. The 19℃ and 28℃ heat stress groups were fed with normal diet, and the 28℃ arginine supplemented group was fed with 600 ppm arginine supplemented diet. Other management measures during the experiment were the same as those during the temporary rearing period. The total length and body weight of juvenile Huso dauricus were measured on the 53rd day. Liver samples were collected after anesthesia in an ice bath and snap-frozen in liquid nitrogen and stored in the refrigerator at -80℃ for further experiments. The reactive oxygen species (ROS) level, the protein expression levels of ferroptosis factors (GPX4 and COX2), the key factor of FoxO1 in the FoxO signaling pathway and the transcriptome expression of juvenile Huso dauricus were measured by fluorescence staining, immunohistochemistry and transcriptome technology. The purpose of this study was to investigate the effect of L-arginine on liver heat stress in Huso dauricus and its molecular mechanism. The results showed that dietary L-arginine could significantly alleviate the heat stress-induced decline in survival and growth performance of juvenile Huso dauricus and effectively reduce the level of reactive oxygen species (ROS) in the liver tissue (p < 0.05). A total of 5854 differentially expressed genes (DEGs) were screened in the liver tissue of juvenile Huso dauricus in the 19℃, 28℃ and 28℃+R groups. These DEGs were mainly enriched in GO terms related to lipid metabolism and amino acid metabolism. KEGG enrichment and immunohistochemical analysis showed that ferroptosis, FoxO signaling pathway and PI3K/AKT signaling pathway were up-regulated by heat stress, while the ferroptosis and FoxO signaling pathway were down-regulated by L-arginine addition. The results of immunohistochemistry showed that the protein expression levels of COX2 and FOXO1 in the 28℃ group were significantly higher than those in the 19℃ group, while the protein expression level of GPX4 was significantly lower than that in the 19℃ group (p < 0.05). Meanwhile, the heat stress-induced changes in protein expression levels were effectively alleviated by the addition of L-arginine. In addition, the expression of PIK3AP1, PIK3R5, and PCK1 was upregulated by heat stress, and the overexpression of these genes was effectively alleviated by L-arginine addition. Our results suggest that Huso dauricus could induce oxidative stress in the liver and further induce ferroptosis in liver cells under heat stress. L-arginine, through its antioxidant effect, can increase the protein expression level of antioxidant enzymes such as GPX4, inhibit FoxO signaling pathway, reduce the accumulation of ROS in liver tissue, and thus alleviate oxidative stress-induced ferroptosis. Meanwhile, L-arginine could alleviate the harmful effects of heat stress on Huso dauricus by inhibiting the over-activation of PI3K-Akt pathway and regulating energy metabolism. Our results not only provide preliminary insights into the protective mechanism of L-arginine on the liver under heat stress conditions, but also provide basic data for the development of anti-stress strategies for healthy sturgeon farming.