Type 2 diabetes has a series of metabolic aberrations accompanied by chronic hyperglycemia, along with various comorbidities. In recent reports, punicalagin from pomegranate has been reported to exert hypoglycemic effects against diabetes. The goal of the current research was to investigate the therapeutic effectiveness and elucidate the mechanisms of punicalagin underlying type 2 diabetes. Type 2 diabetes was induced by a high-fat diet (HFD) combined with streptozotocin (STZ) injection in C57BL/6J mice. Punicalagin was administered daily by oral gavage for 4 weeks. The results indicated that high FBG (fasting blood glucose), dyslipidemia and associated islet, liver and kidney injury were observed in the model group mice. Through metabolomics analysis, it was found that the administration of punicalagin could regulate 24 potential biomarkers and their related metabolic pathways. Moreover, the pathological changes in the liver and kidney were mainly mediated by reducing gluconeogenesis and increasing glycogenesis via stimulation of the PI3K/AKT signaling pathway and regulation of the HMGB-1/TLR4/NF-κB signaling pathway, which simultaneously interrelated to ten main pathological pathways. In addition, we confirmed the positive role of punicalagin in glucosamine-induced HepG2 cells and HG-induced HK-2 cells through related mechanistic studies in vitro. In conclusion, these findings suggested that the multi-effect and multi-target action mode of punicalagin had a significant hypoglycemic effect and a protective effect on diabetes mellitus. Punicalagin might serve as an alternative functional food or as a clinical supplemental therapy for the diabetic population to ameliorate metabolic syndrome.