Vitexin, a natural flavonoid compound identified as apigenin-8-C-b-D-glucopyranoside, has been reported to exhibit antioxidative and anti-inflammatory properties. In this study, we investigated its effect on hypoxia-inducible factor-1a (HIF-1a) in rat pheochromacytoma (PC12), human osteosarcoma (HOS) and human hepatoma (HepG2) cells. Vitexin inhibited HIF-1a in PC12 cells, but not in HOS or HepG2 cells. In addition, it diminished the mRNA levels of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF), smad3, aldolase A, enolase 1, and collagen type III in the PC12 cells. We found that vitexin inhibited the migration of PC12 cells as well as their invasion rates, and it also inhibited tube formation by human umbilical vein endothelium cells (HUVECs). Interestingly, vitexin inhibited the hypoxia-induced activation of c-jun N-terminal kinase (JNK), but not of extracellular-signal regulated protein kinase (ERK), implying that it acts in part via the JNK pathway. Overall, these results suggest the potential use of vitexin as a treatment for diseases such as cancer.