Abstract: Glycinin found in soybean meal has been identified as an antinutrient factor, capable of impeding the growth and health of aquatic animals. However, limited research exists regarding the impact of glycinin on the intestinal microbiota and antimicrobial related genes in red swamp crayfish (Procambarus clarkii). This study aimed to investigate the effects of five different levels of glycinin on intestinal microbiota and antimicrobial related genes of crayfish. Crayfish with an average body weight of approximately 4.3 g subjected to diets containing five graded levels of glycinin (0, 1.82%, 3.64%, 5.46%, and 7.28%) over a period of 4 weeks. The groups were designated based on the level of dietary glycinin as follows: G0 (control group), GL, GH1, GH2, and GH3 group, respectively. Following the feeding trial, alterations and distinctions in the expression of intestinal microbiota and selected genes were evaluated. 16S rRNA gene sequencing showed no significant difference in alpha diversity among the groups. However, the Beta diversity was affected by different levels of dietary glycinin, indicating a shift in the composition of intestinal microbiota. Relative abundance analysis demonstrated a significant increase in Aeromonas sharmana and Cloacibacterium in the GL group compared to other groups. Conversely, Rhodobacter, Paracoccus, Propionicimonas, Hyphomicrobium, and Caulobacter henricii exhibited a significant decrease in abundance of GH2 or GH3 groups compared to the G0 group. Additionally, Agromyce, Microbacterium barkeri, Pseudoxanthomonas, and Legionella were significantly enriched in the GH3 group. In addition, the expression of antimicrobial related genes displayed a complex trend of linear and quadratic responses with increasing levels of dietary glycinin. Initially, all selected genes were down-regulated, followed by up-regulation, and finally down-regulation. Specifically, compared to G0 group, the GL group showed a significant decrease in the expression of alf, cru, and hem. However, with increasing levels of dietary glycinin, the expression of iebf-2, hsp-70, alf, cru and hem, lec-c, and lys in GH1, GH2, and GH3 groups significantly increased. Additionally, the expression of cst-b was significantly enhanced in GH2 group compared to other groups. Spearman correlation analysis demonstrated a significant correlation between the intestinal microbiota and antibacterial related genes. The relative expression levels of iebf-2, hsp-70, alf, cru, hem, lec-c, lys, and cst-b were significantly negatively correlated with the relative abundance of Caulobacter henricii, Paracoccus, Rhodobacter, Propionicimonas, Desulfovibrio putealis, Thermomonas dokdonensis, Aeromonas sharmana, Aeromonas, Chitinilyticum aquatile, Chitinilyticum tainanensis, Cloacibacterium, and Hyphomicrobium, while significantly positively correlated with the relative abundance of Agromyces, Clostridium Perfringens, Microbacterium barkeri, Microbacterium barkeri, Lactococcus, Leuconostoc, Nocardiodes, and Peredibacter starri. In conclusion, to a certain extent, a low level of glycinin in diet (1.82%) could promote the growth of intestinal probiotics and enhance the health of intestinal microbiota, accompanied by down-regulation of antimicrobial-related genes. However, high levels of dietary glycinin (≥3.64%) could inhibit the growth of probiotics while promoting the reproduction of pathogenic bacteria in the intestine, thereby destroying the homeostasis of intestinal microbiota and up-regulating the expression of antimicrobial related genes. The results of correlation analysis suggest potential interactions between antibacterial related genes and intestinal microbiota.