Hydrogen peroxide is an important mediator of intracellular signaling, which potently enhances the expression of heme oxygenase-1 (HO-1) and upregulates synthesis of vascular endothelial growth factor (VEGF). The purpose of the present study was to explore the involvement of HO-1 in regulation of H₂O₂-mediated induction of VEGF synthesis. We provide genetic evidence that basal and H₂O₂-induced VEGF synthesis is partially dependent on HO-1. Inhibition of HO-1 activity by tin protoporphyrin (SnPPIX) resulted in downregulation of VEGF synthesis in murine fibroblasts and human keratinocytes. The relationship between HO-1 and VEGF was corroborated by using cells derived from HO-1 knockout mice, which demonstrated lower basal and H₂O₂-induced production of VEGF. Additionally, knock out of HO-1 gene impaired induction of VEGF by hemin, lysophosphatidylcholine, and prostaglandin-J₂. Our results provide confirmation for the involvement of HO-1 in regulation of angiogenesis.