Abstract: This study aims to investigate the appropriate stocking density for broodstock Macrobrachium rosenbergii. The experiment was conducted in a greenhouse setting, using M. rosenbergii broodstock with a sex ratio of 2﹕1 (females to males). The initial weight of the female shrimps was (17.95±1.11) g, and the male shrimp weighed (21.39±1.63) g. Three stocking densities were tested: LD (1.0 kg/m³), MD (1.5 kg/m³), and HD (2.0 kg/m³). The effects of these stocking densities on brooding rate, survival rate, water quality, and microbial communities in both water and the shrimp gut were analyzed. The results showed that before water quality stabilized, ammonia nitrogen and nitrite nitrogen levels increased with higher stocking density, with the HD group exhibiting significantly higher levels than the LD group (P<0.05). After stabilizing the water quality, no significant differences were observed among the density groups (P>0.05). The ovulation rate of female shrimp in the LD and MD groups was significantly higher than that in the HD group (P<0.05), while there was no significant difference between the LD and MD groups (P>0.05). Survival rate decreased significantly as stocking density increased (P<0.05). Meanwhile, stocking density had a notable effect on the size distribution of broodstock M. rosenbergii, with a significantly higher proportion of smaller shrimp in the higher density groups (P<0.05). Gut microflora results showed that stocking density had no significant effect on bacterial community composition at any sampling time. However, at the peak of ammonia nitrogen content, the proportion of potentially pathogenic Citrobacter increased in both MD and HD groups, indicating that water quality changes caused by higher density significantly influenced the gut microbiota composition in these groups. In conclusion, a stocking density of 1.0 kg/m³ was suggested as the most suitable for broodstock M. rosenbergii. The results of this study provide a valuable reference for determining the appropriate stocking density for M. rosenbergii.