Hydrogen sulfide (H2S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.