The goal of the current study was to investigate the role of exogenous and endogenous hydrogen sulfide (H₂S) on neovascularization and wound healing in vitro and in vivo. Incubation of endothelial cells (ECs) with H₂S enhanced their angiogenic potential, evidenced by accelerated cell growth, migration, and capillary morphogenesis on Matrigel. Treatment of chicken chorioallantoic membranes (CAMS) with H₂S increased vascular length. Exposure of ECs to H₂S resulted in increased phosphorylation of Akt, ERK, and p38. The KATP channel blocker glibenclamide or the p38 inhibitor SB203580 abolished H₂S-induced EC motility. Since glibenclamide inhibited H₂S-triggered p38 phosphorylation, we propose that KATP channels lay upstream of p38 in this process. When CAMs were treated with H₂S biosynthesis inhibitors dl-propylargylglycine or beta-cyano-L-alanine, a reduction in vessel length and branching was observed, indicating that H₂S serves as an endogenous stimulator of the angiogenic response. Stimulation of ECs with vascular endothelial growth factor (VEGF) increased H₂S release, while pharmacological inhibition of H₂S production or KATP channels or silencing of cystathionine gamma-lyase (CSE) attenuated VEGF signaling and migration of ECs. These results implicate endothelial H₂S synthesis in the pro-angiogenic action of VEGF. Aortic rings isolated from CSE knockout mice exhibited markedly reduced microvessel formation in response to VEGF when compared to wild-type littermates. Finally, in vivo, topical administration of H₂S enhanced wound healing in a rat model, while wound healing was delayed in CSE⁻/⁻ mice. We conclude that endogenous and exogenous H₂S stimulates EC-related angiogenic properties through a KATP channel/MAPK pathway.