Hydrogen sulfide (H₂S), like nitric oxide (NO), causes smooth muscle relaxation, but unlike NO, does not stimulate soluble guanylyl cyclase (sGC) activity and generate cGMP. The aim of this study was to investigate the interplay between NO and H₂S in colonic smooth muscle. In colonic smooth muscle from rabbit, mouse and human, L-cysteine, substrate of cystathionine--lyase (CSE), or NaHS, an H₂S donor, inhibited phosphodiesterase 5 (PDE5) activity and augmented the increase in cGMP levels, IP3 receptor phosphorylation at Ser¹⁷⁵⁶ (measured as a proxy for PKG activation), and muscle relaxation in response to NO donor, s-nitrosoglutathione (GSNO), suggesting augmentation of cGMP/PKG pathway by H₂S. The inhibitory effect of L-cysteine, but not NaHS, on PDE5 activity, was blocked in cells transfected with CSE siRNA or treated with CSE inhibitor, DL-propargylglycine (DL-PPG), suggesting activation of CSE and generation of H₂S in response to L-cysteine. H₂S levels were increased in response to L-cysteine and the effect of L-cysteine was augmented by GSNO in a cGMP-dependent protein kinase-sensitive manner, suggesting augmentation of CSE/H₂S by cGMP/PKG pathway. As a result, GSNO-induced relaxation was inhibited by DL-PPG. In flat-sheet preparation of colon, L-cysteine augmented calcitonin-gene-related peptide release in response to mucosal stimulation and in intact segments, L-cysteine increased the velocity of pellet propulsion. These results demonstrate that in colonic smooth muscle, there is a novel interplay between NO and H₂S. NO generates H₂S via cGMP/PKG pathway and H2S, in turn, inhibits PDE5 activity and augments NO-induced cGMP levels. In the intact colon, H₂S promotes colonic transit.