Background and Purpose Hydrogen sulphide (H2S) is synthesised endogenously through cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). Although exogenous H2S is known to produce vasodilatation, the vascular effect of H2S produced through 3-MST is unknown. In this study we demonstrate the effect of a novel inhibitor of 3-MST, DPHE, and determined the effect of this compound on contractile responses in porcine coronary artery. Experimental Approach Synthesis of H2S through 3-MST, and CBS/CSE was determined in rat liver cytosols. Effects of 3-MST inhibitors DPHE, 3-PAB, or I3MT-3, or CBS/CSE inhibitors AOAA and PPG on contractile responses in porcine coronary arteries were determined using isolated tissue baths. Key Results DPHE inhibited the production of H2S from 3-meraptopyruvate (IC50 ~8 µM). The 3-MST inhibitors DPHE, I3MT-3, and 3-PAB all inhibited contractions to U46619 in porcine coronary artery segments through an endothelium-independent mechanism. DPHE and 3MT-3 reduced the U46619 contractions in the absence of extracellular calcium and inhibited the contraction to the L-type calcium channel opener BAY K8644. The combination of AOAA (100 µM) and PPG (10 µM) had no effect on the U46619 contractions. The inhibitory effect of the 3-MST inhibitors does not appear to involve Rho kinase, ERK-MAP kinase, or the mitochondrial electron transport chain. Conclusions and Implications Inhibition of 3-MST in coronary arteries leads to an inhibition of both calcium-dependent and -independent contractions, whereas CBS/CSE inhibitors had no effect on receptor mediated contractions. These data suggest that 3-MST, not CBS/CSE, regulates vascular tone in porcine coronary artery.