Hydrogen sulfide (H2S) is a novel gaseous transmitter, regulating a multitude of biological processes in the cardiovascular and other systems. However, it remains unclear whether it exerts any effect on arterial thrombosis. In this study, we examined the effect of H2S on ferric chloride (FeCl3)-induced thrombosis in the rat common carotid artery (CCA). The results revealed a decrease of the H2S-producing enzyme cystathionine c-lyase (CSE) expression and H2S production that persisted until 48 h after FeCl3 application. Intriguingly, administration with NaHS at appropriate regimen reduced the thrombus formation and enhanced the blood flow, accompanied with the alleviation of CSE and CD31 downregulation, and endothelial cell apoptosis in the rat CCA following FeCl3 application. Moreover, the antithrombotic effect of H2S was also observed in Rose Bengal photochemical model in which the development of thrombosis is contributed by oxidative injury to the endothelium. The in vitro study demonstrated that the mRNA and protein expression of CSE, as well as H2S production, was decreased in hydrogen peroxide (H2O2)-treated endothelial cells. Exogenous supplement of NaHS and CSE overexpression consistently alleviated the increase of cleaved caspase-3 and endothelial cell damage caused by H2O2. Taken together, our findings suggest that endogenous H2S generation in the endothelium may be impaired during arterial thrombosis and that modulation of H2S, either exogenous supplement or boost of endogenous production, may become a potential venue for arterial thrombosis therapy.