The purpose of the present study is to investigate the effect of L-cysteine on colonic motility and the underlying mechanism. Immunohistochemical staining and Western blot were used to detect the localization of the H2S-generating enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Organ bath system was used to observe the muscle contractile activities. Whole-cell patch-clamp technique was applied to record ionic channels currents in colonic smooth muscle cells. The results showed that both CBS and CSE were localized in mucosa, longitudinal and circular muscle and enteric neurons. L-cysteine had a dual effect on colonic contraction, and the excitatory effect was blocked by pretreatment with CBS inhibitor aminooxyacetate acid (AOAA) and CSE inhibitor propargylglycine (PAG); L-cysteine concentration-dependently inhibited L-type calcium channel current (ICa,L) without changing the characteristic of L-type calcium channel (P < 0.01); In contrast, the exogenous H2S donor NaHS increased ICa,L at concentration of 100 μmol/L, but inhibited ICa,L and modified the channel characteristics at concentration of 300 μmol/L (P < 0.05); Furthermore, L-cysteine had no effect on large conductance calcium channel current (IBKCa), but NaHS significantly inhibited IBKCa (P < 0.05). These results suggest that L-cysteine has a potential dual effect on colonic smooth muscle and the inhibitory effect might be directly mediated by L-type calcium channel while the excitatory effect might be mediated by endogenous H2S.