Genome-wide screening using a small interfering RNA (siRNA) library has revealed novel molecules that are involved in a wide range of physiological responses. The expression of vascular endothelial growth factor (VEGF) is increased under hypoxic conditions, and plays a crucial role in tumor angiogenesis and tissue responses to ischemia. Here, we used a siRNA expression vector library to elucidate molecules that modify VEGF expression. Screening using an siRNA library revealed that MAPKKK6 (MEKK6/MAP3K6) regulates VEGF expression under both normoxic and hypoxic conditions in vitro, although the biological function of MAP3K6 remains unknown. Attenuation of VEGF expression by MAP3K6 inhibition was demonstrated by transient transfection of double-stranded RNA as well as by stable transfection of short hairpin RNA-expressing vectors against MAP3K6. Conditioned medium of MAP3K6-knocked down cells attenuated both endothelial proliferation and capillary network formation in a VEGF-dependent manner in vitro. In addition, tumor cells with down-regulation of MAP3K6 expression showed significant suppression of tumor growth in vivo, which was accompanied by significant repression of vessel formation and VEGF expression in these tumors. The results of this study suggest that MAP3K6 regulates VEGF expression in both normoxia and hypoxia, and that regulation of VEGF by MAP3K6 may play a crucial role in both angiogenesis and tumorigenesis.