Peritoneal fibrosis (PS) determines the long-term outcome of peritoneal dialysis (PD). We previous confirmed that hydrogen sulfide (H2S) inhibited PS, but its cellular mechanism was not fully elucidated. Epithelial-mesenchymal transition (EMT) of mesothelial cells (MCs) is an important cellular event of PS, we therefore investigated whether EMT can be affected by H2S in MCs. Rats were treated with 4.25% -glucose PD fluids plus lipopolysaccharide for 28 days to produce PS, and NaHS (56 μg/kg.d) was given simultaneously. NaHS (56 μg/kg.d) reduced the deposition of collagen in the submesothelial zone compared with the PS group. In primarily cultured rat MCs, 4.25% -glucose PD fluid induced EMT in MCs featured as loss of ZO-1 and Cytokeratin, and increase of α-SMA, plasminogen activator inhibitor 1, fibronectin and TGF-β1 proteins. PD fluid also increased IL-6 and monocyte chemotactic protein-1 mRNA expressions as well as the phosphorylation of Smad2/3 and Smad3. NaHS (50-300 μmol/L) reversed the above alterations with the optimal dose at 100 μmol/L. Thus, exogenous H2S improves PS by inhibiting EMT in MCs. The anti-EMT effect of H2S is associated with the inhibition of inflammation and TGF-β1-Smad signal pathway.