This study investigates the inflammatory response to intra-plantar injection of L -cysteine in a murine model. L -cysteine induces a two-phase response: an early phase lasting 6 h and a late phase peaking at 24 h and declining by 192 h. The early phase shows increased neutrophil accumulation at 2 h up to 24 h, followed by a reduction at 48 h. On the other hand, the late phase exhibits increased macrophage infiltration peaking at 96 h. Inhibition of cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway, significantly reduces L -cysteine-induced edema, suggesting its dependence on CBS-derived hydrogen sulfide (H ₂ S). Sequential formation of sphingosine-1-phosphate (S1P) preceding nitric oxide (NO) generation suggests the involvement of a CBS/S1P/NO axis in the inflammatory response. Inhibition of de novo sphingolipid biosynthesis, S1P1 receptor, and endothelial NO synthase (eNOS) attenuates L -cysteine-induced paw edema. These findings indicate a critical role of the CBS/H ₂ S/S1P/NO signaling pathway in the development and maintenance of L -cysteine-induced inflammation. The co-presence of H ₂ S and NO is necessary for inducing and sustaining the inflammatory response, as NaHS or L -arginine alone do not replicate the marked and prolonged inflammatory effect observed with L -cysteine. This study enhances our understanding of the complex molecular mechanisms of the interplay between NO and H ₂ S pathways in inflammation and identifies potential therapeutic targets for inflammatory disorders.