Abstract: The study investigated the hypoglycemic activity and mechanism of a combination comprising wheat peptides and mulberry leaf extract. In alloxan-glucose co-induced hyperglycemic zebrafish models, the 1:2 (w/w) combination exhibited hypoglycemic effects. In high-fat diet-induced obese hyperglycemic mice, the combination significantly inhibited body weight gain and blood glucose level, improved glucose metabolism and insulin sensitivity, and alleviated inflammatory responses. Mechanism studies revealed that the combination remodeled gut microbiota composition in obese hyperglycemic mice, decreasing the relative abundance of pathogenic Ileibacterium while increasing probiotics such as Faecalibaculum and Dubosiella. Non-targeted serum metabolomics identified 8 different metabolites, including LysoPC(14:0/0:0), Prostaglandin F2α, and 5-Hydroxyindoleacetate. Correlation analysis demonstrated significant relationships between the relative abundance of gut microbiota (Ileibacterium, Faecalibaculum, Dubosiella, Clostridium_sensu_stricto_1, Coriobacteriaceae_UCG-002) and alterations in these metabolites. Collectively, the wheat peptides and mulberry leaf extract combination potentially exerts hypoglycemic effects through multi-target regulation of glucose-lipid metabolism and inflammation via gut microbiota and serum metabolites, providing new insights for functional food development.