Accumulating evidence shows that hydrogen sulfide (H2S) has physiological functions in various tissues and organs. It includes regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney, and brain from ischemic insult. H2S is produced from L-cysteine by pyridoxal 5'-phosphate (PLP)-dependent enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). 3-Mercaptopyruvate sulfurtransferase (3MST) is the third H2S-producing enzyme. 3-Mercaptopyruvate (3MP) is provided from L-cysteine and α-ketoglutarate by a PLP-dependent cysteine aminotransferase (CAT). An additional pathway for the production of H2S from D-cysteine involving 3MST and D-amino acid oxidase (DAO) has been identified. Recently, hydrogen polysulfides (H2Sn), which are oxidized forms of H2S, have been found to stimulate transient receptor potential ankyrin1 (TRPA1) channel, much more potently than H2S. H2Sn is produced from 3MP by 3MST. In addition to the enzymatic production, the interaction between H2S and nitric oxide (NO), another gaseous signaling molecule, also generates H2Sn. These observations provide new insight into the production of these molecules and their mechanisms of physiological functions.