Hydrogen sulfide has emerged as an important endothelium-dependent vasodilator, but its role in shear stress-mediated dilation of coronary arteries is unclear. We examined the role of H₂S on shear stress-mediated dilation of isolated mouse coronary arteries. In these vessels, Na₂S produced concentration-dependent dilation, which was significantly inhibited by iberiotoxin and by 4-aminopyridine. In addition, BK and Kv currents in mouse coronary smooth muscle cells were directly activated by Na₂S, suggesting that H₂S produced vasodilation through BK and Kv channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm²) and produced graded dilatory responses, but such effects were diminished in the presence of 100 μM Na₂S. Pre-incubation with the cystathionine γ-lyase inhibitor, d,l-propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress-mediated vasodilation. However, in the presence of L-NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress-mediated vasodilation, suggesting an interaction between NO and H₂S signaling. Na₂S inhibited eNOS activity in cultured mouse aortic endothelial cells and reduced the level of phospho-eNOS(serine 1177). These results suggest that both NO and H₂S are important shear stress-mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H₂S through inhibition of NO generation.