Hydrogen sulfide (H2S), which was considered as a novel gasotransmitter, is produced endogenously from L-cysteine in mammalian brain and vessels, and might be a physiological function regulator to these organs. Here, we showed that mRNA for H2S producing enzyme, cystathionine gamma-lyase, was expressed in myocardial tissues and H2S could endogenously be produced in myocardial tissues. Negative inotropic effect of H2S was proved in present study in vitro and in vivo experiments, and the effect could partly be blocked by glibenclamide, a KATP channel blocker. An intravenous bolus injection of NaHS provoked a decrease in central venous pressure. The present findings suggested that H2S could be endogenously produced by heart tissues, as a physiological cardiac function regulator, mediated by KATP channel pathway.