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This study was to investigate the anti-diabetic effects and molecular mechanisms of Tang-Kang-Fu-San (TKFS), a traditional Tibetan medicine, in treating type 2 diabetes mellitus of spontaneous diabetic db/db mice. Firstly HPLC fingerprint analysis was performed to gain the features of the chemical compositions of TKFS. Next different doses of TKFS (0.5 g/kg, 1.0 g/kg, and 2.0 g/kg) were administrated via oral gavage to db/db mice and their controls for 4 weeks. TKFS significantly lowered hyperglycemia and ameliorated insulin resistance (IR) in db/db mice, indicated by results from multiple tests, including fasting blood glucose test, intraperitoneal insulin and glucose tolerance tests, fasting serum insulin levels and homeostasis model assessment of IR analysis as well as histology of pancreas islets. TKFS also decreased concentrations of serum triglyceride, total and low-density lipoprotein cholesterol, even though it did not change the mouse body weights. Results from western blot and immunohistochemistry analysis indicated that TKFS reversed the down-regulation of p-Akt and p-AMPK, and increased the translocation of Glucose transporter type 4 in skeletal muscles of db/db mice. In all, TKFS had promising benefits in maintaining the glucose homeostasis and reducing IR. The underlying molecular mechanisms are related to promote Akt and AMPK activation and Glucose transporter type 4 translocation in skeletal muscles. Our work showed that multicomponent Tibetan medicine TKFS acted synergistically on multiple molecular targets and signaling pathways to treat type 2 diabetes mellitus.