Background and Aims Acute pancreatitis (AP) accompanied by intestinal injury and intestinal barrier dysfunction can significantly worsen AP prognosis. Currently, there is no effective clinical treatment for AP-related intestinal dysfunction. Ulinastatin (UTI) is a conventional drug used for AP treatment due to its ability to inhibit trypsin activity and exert anti-inflammatory effects. However, whether UTI directly improves AP-related intestinal injury remains unclear. Therefore, this study was conducted to investigate the therapeutic effects and potential mechanisms of UTI in AP-related intestinal dysfunction.Methods Thirty rats were equally randomized into three groups and received intraperitoneal injections of PBS (control group), 20% L-arginine (AP group), or 20% L-arginine + UTI (UTI group). Tail vein blood samples were collected at 0, 24, and 48 h after the initial injection of L-arginine (or PBS), and the rats were euthanized after the final blood collection to obtain pancreatic and terminal ileum tissues. Serum levels of amylase, lipase, intestinal fatty acid-binding protein (I-FABP), and diamine oxidase (DAO) were measured using ELISA. Histopathological examinations of the pancreas and terminal ileum were conducted. Western blot was used to detect the protein expression levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), and interleukin 10 (IL-10) in terminal ileum tissue. Western blot and qRT-PCR were used to assess the protein and mRNA expression levels of Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) p-p65 in the terminal ileum.Results Compared with the control group, both the AP and UTI groups exhibited significant AP changes (elevated serum amylase and lipase levels, pancreatic histopathological damage) and AP-related intestinal injury (decreased I-FABP and DAO levels, ileal histopathological damage). However, these alterations were significantly less severe in the UTI group compared to the AP group (all P<0.01). Compared with the control group, both the AP and UTI groups showed significantly increased protein expression of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and the anti-inflammatory factor IL-10 in the ileal tissue; however, the UTI group exhibited significantly lower levels of pro-inflammatory factors and higher levels of IL-10 compared to the AP group (all P<0.01). Additionally, compared with the control group, both the AP and UTI groups displayed significant upregulation of TLR4 and NF-κB p-p65 protein and mRNA expressions in ileal tissue, but the upregulations were significantly lower in the UTI group compared to the AP group (all P<0.01).Conclusion UTI can inhibit the activation of the TLR4/NF-κB signaling pathway in the ileal tissue of AP rats, thereby reducing pro-inflammatory cytokine levels and increasing anti-inflammatory cytokine levels. Therefore, in addition to its anti-pancreatitis effects, UTI may have a direct therapeutic effect on AP-related intestinal dysfunction.