Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, commonly used in the oral treatment for erectile dysfunction, relaxes smooth muscle of human bladder through the activation of hydrogen sulfide (H2S) signaling. H2S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from l-cysteine (l-Cys) by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Sildenafil also relaxes rat and human myometrium during preterm labor but the underlying mechanism is still unclear. In the present study we investigated the possible involvement of H2S as a mediator of sildenafil-induced effect in uterine mouse contractility. We firstly demonstrated that both enzymes, CBS and CSE were expressed, and able to convert l-Cys into H2S in mouse uterus. Thereafter, sildenafil significantly increased H2S production in mouse uterus and this effect was abrogated by CBS or CSE inhibition. In parallel, l-Cys, sodium hydrogen sulfide or sildenafil but not d-Cys reduced spontaneous uterus contractility in a functional study. The blockage of CBS and CSE reduced this latter effect even if a major role for CSE than CBS was observed. This data was strongly confirmed by using CSE−/− mice. Indeed, the increase in H2S production mediated by l-Cys or by sildenafil was not found in CSE−/− mice. Besides, the effect of H2S or sildenafil on spontaneous contractility was reduced in CSE−/− mice. A decisive proof for the involvement of H2S signaling in sildenafil effect in mice uterus was given by the measurement of cGMP. Sildenafil increased cGMP level that was significantly reduced by CSE inhibition. In conclusion, l-Cys/CSE/H2S signaling modulates the mouse uterus motility and the sildenafil effect. Therefore the study may open different therapeutical approaches for the management of the uterus abnormal contractility disorders.