Methotrexate (MTX) is a commonly used anticancer and immunosuppressive agent. However, MTX can induce hepatotoxicity due to oxidative stress, inflammation, and apoptosis. Hydrogen sulfide (H2S), the endogenous gaseous molecule, has antioxidant, anti-inflammatory, and anti-apoptotic effects. The present work explored the probable protective effect of H2S against MTX hepatotoxicity in rats and also the possible mechanisms underlying this effect. MTX was given at a single intraperitoneal (i.p.) dose of 20 mg/kg. Sodium H2S (56 µmol /kg/day, i.p.), as H2S donor, was given for 10 days, starting 6 days before MTX administration. H2S significantly reduced serum alanine aminotransferase, hepatic malondialdehyde, interleukin 6, nuclear factor κB p65, cytosolic cytochrome c, phosphorylated signal transducer and activator of transcription 3, and Bax/Bcl-2 ratio and significantly increased hepatic total antioxidant capacity and endothelial nitric oxide synthase (eNOS) in rats received MTX. In addition, H2S minimized the histopathological injury and significantly decreased the expression of STAT3 in liver tissue of MTX-challenged rats. The effects of H2S were significantly antagonized by administration of glibenclamide as KATP channel blocker, Nω-nitro-l-arginine, as eNOS inhibitor, or ruthenium red, as transient receptor potential vanilloid 1 (TRPV1) antagonist. It was concluded that H2S provided significant hepatoprotection in MTX-challenged rats through its antioxidant, anti-inflammatory, anti-apoptotic effects. These effects are most probably mediated by the ability of H2S to act as IL-6/STAT3 pathway modulator, KATP channel opener, eNOS activator, and TRPV1 agonist.