Abstract Hydrogen sulfide (H 2 S) plays important roles in metabolism and health. Its enzymatic generation from sulfur-containing amino acids (SAAs) is well characterized. However, the existence of non-enzymatic H 2 S production from SAAs, the chemical mechanism, and its biological implications remain unclear. Here we present non-enzymatic H 2 S production in vitro and in blood via a reaction specific for the SAA cysteine serving as substrate and requires coordinated catalysis by Vitamin B 6 , pyridoxal(phosphate), and iron under physiological conditions. An initial cysteine-aldimine is formed by nucleophilic attack of the cysteine amino group to the pyridoxal(phosphate) aldehyde group. Free or heme-bound iron drives the formation of a cysteine-quinonoid, thiol group elimination, and hydrolysis of the desulfurated aldimine back to pyridoxal(phosphate). The reaction ultimately produces pyruvate, NH 3 , and H 2 S. This work highlights enzymatic production is inducible and robust in select tissues, whereas iron-catalyzed production contributes underappreciated basal H 2 S systemically with pathophysiological implications in hemolytic, iron overload, and hemorrhagic disorders.