Abstract: This study took hydrolyzed peptides from Pelodiscus sinensis as the research object, aiming to investigate their impact on intestinal flora and immune function. Utilizing an in vitro intestinal simulation system and a Caco-2 cell model, 16S rRNA (V3-V4) high-throughput sequencing technology was employed to evaluate the diversity and composition of the intestinal microbiota. Additionally, HPLC was used to quantify the short-chain fatty acid content in the fermentation broth, and qPCR was used to assess the immune factors of Caco-2 cells. The findings revealed that the treatment of P. sinensis hydrolyzed peptides could significantly (P<0.05) decrease the relative abundance of Dialister, Ruminococcus gnavus, and Sutterella in the gut. Furthermore, these peptides enhanced the production of short-chain fatty acids, particularly butyric acid and isovaleric acid, by intestinal microorganisms. Notably, low concentration (5 mg/mL) of P. sinensis hydrolyzed peptides showed a more consistent promotion of short-chain fatty acid production by intestinal microbes compared to the medium (25 mg/mL) and high concentrations (50 mg/mL). Short-chain fatty acids also played a pivotal role in modulating intestinal immune function. The Caco-2 cell model demonstrated that P. sinensis hydrolyzed peptides could regulate the expression of immune-related genes, including TLR4, NF-κB, and MD2, in normal Caco-2 cells. This led to an increase in the expression of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and the anti-inflammatory cytokine IL-10, thereby activating cellular immunity. In an inflammatory cellular model, P. sinensis hydrolysate downregulated the expression of pro-inflammatory cytokines (IL-1β) and upregulated anti-inflammatory cytokine IL-10, while also reducing iNOS relative expression level, thereby alleviating the inflammatory response. In conclusion, P. sinensis hydrolyzed peptides can act as prebiotics by modulating the composition to maintain intestinal homeostasis, and enhancing immune function of the intestinal flora, providing valuable insights into the interaction between animal-derived bioactive peptides and intestinal microorganisms, as well as contributing to the nutritional value evaluation system.