Hypochlorous acid (HOCl) is a strong oxidant produced by myeloperoxidase. Previous work suggested that HOCl modifies the corrin ring of cobalamins to yield chlorinated species via mechanisms that are incompletely understood. Herein, we report a mechanistic study on the reaction between cyanocobalamin (CNCbl, vitamin B12) and HOCl. Under weakly acidic, neutral and weakly alkaline conditions, the reaction produces the c-lactone derivative of CNCbl chlorinated at the C10-position of corrin ring (C10–Cl–CNCbl-c-lactone). Formation of C10–Cl–CNCbl-c-lactone was not observed at pH ≥ 9.9. The chlorination of CNCbl by HOCl proceeds via two pathways involving one and two HOCl molecules: the reaction is initiated by the very fast formation of a complex between CNCbl and HOCl, which either undergoes slow transformation to chlorinated species, or rapidly reacts with a second HOCl molecule to produce C10–Cl–CNCbl. Subsequent reaction of C10–Cl–CNCbl with HOCl proceeds rapidly toward lactone ring formation by H-atom abstraction at position C8. This work uncovered mechanisms and products of the reaction of a biologically active and therapeutically used cobalamin, CNCbl and the endogenous oxidant HOCl. Binding and reactivity studies of C10–Cl–CNCbl and C10–Cl–CNCbl-c-lactone with relevant proteins of the cobalamin pathway and with cultured cells are necessary to elucidate the potential physiological effects of these species.