Introduction <p>While it has been established that intestinal health and microbial variations can influence pancreatic well-being, the extent of intestinal damage and the composition and diversity of the microbiota in animal models of <span>l</span>-arginine-induced acute pancreatitis (AP) remain unclear.</p> Methods <p>This study aims to evaluate the extent of intestinal damage and microbial changes in AP mouse models. The male C57BL/6 mice were randomly divided into two groups: the AP group received an intraperitoneal injection of 10% <span>l-</span>arginine, while the control group received an equivalent volume of saline. Histological analysis was used to assess morphological changes and damage in the pancreas and the duodenum, ileum, and jejunum. Fluorescent immunohistochemistry and immunohistochemical techniques were employed to evaluate the expression of myeloperoxidase (MPO) in the pancreas and Zonula occludens-1 (ZO-1) and Occludin in the small intestine. High-throughput sequencing was utilized to analyze the composition and diversity of the intestinal microbiota.</p> Results <p><span>l-</span>arginine-induced AP in mice significantly increased serum lipase activity and decreased calcium levels, with an increase in pancreatic MPO expression. Additionally, we observed varying degrees of intestinal villi necrosis, edema, and inflammatory cell infiltration in the duodenum, jejunum, and ileum of the AP group mice. Notably, the expression of epithelial Occludin and ZO-1 was reduced in these regions. Compared to healthy mice, AP mice exhibited reduced bacterial richness and diversity, with changes in the intestinal microbiota including increased prevalence of <i>Bacteroidetes</i> and <i>Proteobacteria</i>, while the abundance of <i>Firmicutes</i> decreased.</p> Discussion <p><span>l-</span>arginine-induced acute pancreatitis in mice resulted in significant alterations in the intestinal barrier and microbiota.</p>