Abstract: Objective: This study aimed to investigate the effects of salmon (Oncorhynchus keta) skin protein hydrolysate with high dipeptidyl peptidase IV (DPP-IV) inhibitory activity on blood glucose regulation and intestinal flora in type 2 diabetes mellitus (T2DM) mice. Methods: C57BL/6J mice were randomly divided into a normal control group, a model group, and low- and high-dose salmon skin protein hydrolysate treatment groups. T2DM was induced in the latter three groups via a high-fat diet combined with streptozotocin (STZ) injection. After 28 days of gastric gavage treatment, body weight, food intake, fasting blood glucose level and oral glucose tolerance (OGTT) were measured. Serum fasting insulin content (FINS), homeostasis model assessment of insulin resistance (HOMA-IR), and glucagon-like peptide 1 (GLP-1) levels were analyzed. The organ indicators and histopathological changes of liver and pancreatic islets were evaluated. The structure of intestinal flora in fecal samples was characterized using 16S rDNA sequencing. Results: Compared to the model group, salmon skin protein hydrolysate group treatment could alleviate weight loss, and extremely significantly reduce food intake (P<0.01).It also extremely significantly decreased fasting blood glucose level (P<0.01) in T2DM mice, with a 34.65% reduction in the high-dose group. The high-dose treatment of hydrolysate also extremely significantly down-regulated the levels of FINS, HOMA-IR (P<0.01) and OGTT-AUC (P<0.01), while up-regulated GLP-1 levels (P<0.01). Moreover, it significantly decreased liver and pancreatic organ indicators (P<0.05), improved islet cell morphology, and reduced hepatic vacuolar degeneration. Furthermore, salmon skin protein hydrolysate increased the richness and diversity of intestinal flora, improved community evenness, and elevated the relative abundance of probiotic bacteria in T2DM mice. Conclusion: The high-dose salmon skin protein hydrolysate group (less than 3 kDa) promoted GLP-1 secretion, improved insulin function, effectively reduced FBG, and mitigated hepatic and pancreatic damage in T2DM mice. Furthermore, intestinal flora composition in T2DM mice were partially restored by salmon skin hydrolysate.