To search for bioactive gypenosides and their analogues, a saponin enriched fraction and its hydrolyzate from Gynostemma pentaphyllum were phytochemically investigated. Fractionation by diverse chromatographic methods, including HPLC, Sephadex LH-20, silica gel, and C18 reverse phase silica gel, led to the isolation and purification of twelve triterpenes, including five undescribed and seven known. The chemical structures of all compounds were determined as analyzed by nuclear magnetic resonance (NMR), high resolution mass spectrometry (HR-MS), infrared spectrum (IR), optical rotation, and chemical transformations. Among all isolates, nine compounds possessed a rare dammarane triterpenoid framework with A-ring modified. The relative configurations of three compounds were determined by 2D NMR for the first time. The absolute configurations of four compounds were determined by the modified Mosher's method. Two of all isolated compounds significantly enhanced 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake and Glucose Transporter 4 (GLUT4) translocation via activating the AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) signaling pathway. This study provided the potential candidates for the development of antidiabetic agents.