Ginsenoside Rk3 (G-Rk3) is a main active ingredient of ginsenosides. Several recent studies demonstrated that ginsenosides have potential anti-type 2 diabetes mellitus (T2DM) properties. To evaluate the anti-T2DM effect of G-Rk3 and verify its potential mechanism, a high-fat-diet/streptozocin (HFD/STZ) induced model of T2DM in C57BL/6 mice and a high glucose induced insulin resistance model of HepG2 cells were applied in this research. Our analysis indicated that G-Rk3 reduced HFD/STZ induced hyperglycemia, and serum insulin and inflammation levels, and ameliorated glucose tolerance and insulin resistance, and prevented liver histological changes. Furthermore, it also significantly reduced lipid accumulation as shown by lower TG, LDL-C and TC serum concentrations and Oil Red O staining in liver tissues. The hypoglycemic effect of G-Rk3 seemed to be partially mediated via the inhibition of hepatic gluconeogenesis, which was supported by the activated p-Akt, p-FoxO1 and GLUT2 and inhibited FoxO1, PEPCK and G6pase protein expressions in the liver as well as increased glucose uptake in high glucose induced HepG2 cells. The gene expressions of hepatic gluconeogenesis were also down-regulated by G-Rk3 in HFD/STZ induced T2DM mice. In addition, G-Rk3 suppressed HFD/STZ induced lipid accumulation by regulating related gene and protein expressions such as p-ACC, FAS and SREBP-1, which are the downstream targets of AMPK. AMPK and Akt inhibitors significantly reversed G-Rk3 mediated hepatic gluconeogenesis and lipid accumulation. Thus, our study is the first to illustrate that G-Rk3 mediates hepatic gluconeogenesis and lipid accumulation via activating the AMPK/Akt signaling pathway in HFD/STZ induced T2DM mice.