Vitamin K4 (VK4) is a synthetic hydrophilic menadione compound, which is clinically used as hemostasis medicine. It has been reported that several vitamin Ks had inhibitory effects on various cancer cells. However, there is no report about VK4s anticancer activity. The goal of this study was to investigate the inhibitory effect of VK4 on human prostate PC-3 cells and the mechanisms involved. We found that VK4 dose-dependently inhibited cell proliferation in PC-3 cells with an IC50 value of about 20.94 microM. Hoechst 33258 Staining results showed that VK4 caused DNA fragmentation in PC-3 cells. PI staining results indicated that VK4-induced PC-3 cell cycle arrest at the S phase. Further mechanistic studies revealed that VK4-mediated induction of apoptosis in PC-3 cell is associated with disruption of mitochondrial membrane potential, down-regulation of Bcl-2, and up-regulation of Bax, release of cytochrome c from mitochondria, and activation of caspase-3 and PARR Thus, VK4 might be useful in prostate cancer chemotherapy.