Momordica charantia (MC) has been used as an alternative therapy for diabetes mellitus. This study analyzed and elucidated therapeutic targets contributing to the hypoglycemic effect of aqueous extract of MC seeds (MCSE) by transcriptomic analysis. Protein ingredients aimed at the hypoglycemic target were further identified by proteomic, docking, and receptor-binding assays. The data showed that MSCE (1 g/kg) significantly lowered the blood glucose level in normal and diabetic mice. Moreover, MCSE primarily regulated the insulin signaling pathway in muscles and adipose tissues, suggesting that MCSE might target insulin receptor (IR), stimulate the IR-downstream pathway, and subsequently display hypoglycemic activity in mice. It was further revealed that inhibitor against trypsin (TI) of MC directly docked into IR and activated the kinase activity of IR in a dose-dependent manner. In conclusion, the findings suggested that MCSE regulated glucose metabolism mainly via the insulin signaling pathway. Moreover, TI was newly identified as a novel IR-binding protein of MC that triggered the insulin signaling pathway via binding to IR.