In eukaryotes, hydrogen sulfide (H₂S) acts as a signaling molecule and cytoprotectant. It modulates synaptic transmission, relaxes smooth muscle, and regulates a release of insulin, endoplasmic reticulum stress and apoptosis. H₂S also protects neurons and cardiac muscle from oxidative stress and ischemia reperfusion injury. H₂S is known to be produced from L-cysteine by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) coupled with cysteine aminotransferase (CAT). Here we report an additional biosynthetic pathway for the production of H₂S from D-cysteine involving 3MST and D-amino acid oxidase (DAO). Unlike the L-cysteine pathway, this D-cysteine-dependent pathway seems to operate predominantly in the cerebellum and the kidney. Our study revealed that administration of D-cysteine protected primary cultures of cerebellar neurons from oxidative stress induced by H₂O₂ and attenuated ischemia-reperfusion injury in the kidney more than L-cysteine. This study presents a novel pathway of H₂S production and provides a new therapeutic approach to deliver H₂S to specific tissues.