Hydrogen sulfide (H2S) is substantially converted from cysteine by the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). H2S can profoundly affect most organ systems in animals and humans by inducing a wide range of physiological functions. However, the roles of H2S in the progression of endometriosis remain unknown. The aim of the current study was to test the hypothesis that H2S might play a role in the pathogenesis of endometriosis via modulating the biological behavior of endometrial stromal cells (ESCs). First, we explored the expression level of CBS and CSE in ESCs via immunohistochemistry and immunocytochemistry. Second, cell Count Kit-8 (CCK-8) assays were utilized to investigate the cell viability of human ESCs (HESCs) in vitro. Third, we studied the potential effects of H2S in a rodent model of endometriosis. Both CBS and CSE were overexpressed in endometriotic lesions. Exogenous and endogenous H2S could promote HESC proliferation in vitro. Furthermore, this pro-proliferation effect could be reversed by treating with inhibitors of CBS, CSE, or the NF-kappa B pathway. In vivo, we uncovered that inhibitors of CBS and CSE could remarkably reduce the number and weight of mouse endometriotic lesions. These data suggested that H2S promotes ESC proliferation via activation of the NF-kappa B pathway, which provides a scientific basis for the clinical application of blocking H2S to treat endometriosis.