Introduction Hydrogen sulfide (H2S) is generated in mammalian cells mainly by one of the two pyridoxal-5′-phosphate-dependent enzymes, cystathione-γ-lyase (CSE), and cystathione-β-synthase (CBS) using L-cysteine as the main substrate. In previous studies, we found that CBS and CSE were functionally expressed in vas deferens (VD) and H2S-mediated VD smooth muscle relaxation. However, the detail mechanisms that H2S-relaxed VD smooth muscle were unknown so far. Aim The aim of this study is to explore the molecular target sites of H2S in VD smooth muscle. Methods Isolated rat VD smooth muscle strips were used for tension recording in vitro. Double immunofluorescence staining was used to identify the localization of large conductance Ca2+-activated K+ (BKCa) channels. Main Outcome Measures Changes in tonic contraction of isolated rat VD smooth muscle strip were measured after the treatment of drugs. The expression of BKca channels in rat VD smooth muscle cells was also assessed. Results The results showed that L-NG-nitroarginine methyl ester (a nitric oxide synthase inhibitor) did not affect the response of VD to sodium hydrosulphide (NaHS), suggesting that nitric oxide pathway was not involved. Further studies revealed that transient receptor potential (TRP) channels did not contribute to the NaHS-induced relaxant effect. Glibenclamide, an ATP-sensitive K channel blocker, did the same thing, whereas BKCa channel blockers iberiotoxin or tetraethylammonium largely reversed the relaxant effect, suggesting that H2S may target BKCa channels. We also confirmed that BKCa channels were localized in VD smooth muscle cells. Then, studies revealed that NaHS-induced VD smooth muscle relaxation was abolished by N-ethylmaleimide, which was widely used as a sulfhydryl alkylation compound protecting thiols from oxidation, whereas DL-Dithiothreitol, a strong reducing agent, did not affect the response of VD to NaHS. Conclusions We concluded that H2S relaxed the VD smooth muscle by targeting BKCa channels via redox-mediated mechanism.