Abstract: The study was carried out to investigate the optimal stocking density of Penaeus monodon. Healthy shrimps initial body weight (2.55±0.19) g, body length (6.71±0.20) cm were randomly distributed into six different stocking densities (225, 300, 375, 450, 525, and 600 ind/m³), labeled as D225, D300, D375, D450, D525, and D600, respectively. Culturing for 42 days. The results showed that the survival rate (SR) gradually decreased with increasing stocking density. Furthermore, the weight gain rate (WGR), specific growth rate (SGR), and condition factor (CF) were increased first and then decreased, while the feed conversion ratio (FCR) was conversely. Antioxidant and immune properties of hepatopancreatic indicated that D375 showed the best antioxidant and immune performance, whose activities of SOD, AKP, and ACP were significantly higher than that of the other groups (P<0.05), and the MDA content and expression of Prx, p53, MyD88, and LZM were significantly lower than that of the other groups (P<0.05). Regarding intestinal health, the Alpha diversity index of intestinal microbiota showed a trend to increase first and then decrease with the increasing stocking density. The Alpha diversity of D225 and D600 was significantly lower than that of the other groups (P<0.05). As for species distribution of intestinal microbiota, D450 showed the highest relative abundance of beneficial bacteria (e.g., Firmicutes and Ligilactobacillus) and the lowest one of harmful bacteria (e.g., Proteobacteria). By observing the intestinal structure and morphology, it was found that the intestinal structure of D375 and D450 was more complete, the villi arranged densely and neatly, and the villi height was higher than that of the other groups. In contrast, the other groups exhibited partial epithelial cell sloughing and villus loss. In summary, the study indicates that excessively high or low stocking density was not conducive to shrimp growth and physiological function regulation. The stocking density of 375-450 ind/m³ promoted growth, improved hepatopancreas antioxidant capacity and non-specific immunity and optimized the intestinal microbial community structure of the shrimp. This study provided an experimental basis for the optimum culturing density of P. monodon, which could help to improve the culture efficiency and promote the development of sustainable and healthy shrimp aquaculture.