Vasculogenic progenitor cells (VPCs) circulate in the blood and have the ability to differentiate into endothelial cells that make up the lining of blood vessels. Therefore, VPC transplantation is a new strategy for the treatment of ischemic diseases. Because priming/preconditioning of VPCs before transplantation enhances their regenerative potential, the present study investigated whether ent-16α,17-dihydroxy-kauran-19-oic acid (DHK) isolated from Siegesbeckia pubescens could stimulate/activate VPCs in vitro. Therefore, the effect of DHK (1-100 µM concentration) on the proliferation, migration, and tube forming of VPCs was examined in various systems, and related signaling pathways were identified. DHK treatment significantly increased the proliferation, migration, and tube formation of VPCs in a dose-dependent manner. Phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and Akt was significantly increased by DHK, but chemical inhibitors against ERK1/2 (U0126) and Akt (LY294002) significantly attenuated DHK-enhanced proliferation, migration, and tube formation of VPCs. Collectively, these results indicated that DHK shows promise as a novel VPC primer/activator.