Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with cell death in the myocardium. In this study, we investigated potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT-sensitive homodimer which increases after myocardial ischemia/reperfusion (I/R). The presence of the antioxidant N-acetylcysteine reduces I/R-induced homodimerization of Bnip3. Overexpression of Bnip3 in cells revealed that most of exogenous Bnip3 exists as a DTT-sensitive homodimer which correlates with increased cell death. In contrast, endogenous Bnip3 exists mainly as a monomer under normal conditions in the heart. Screening of the Bnip3 protein sequence revealed a single conserved cysteine residue at position 64. Mutation of this cysteine to an alanine (Bnip3C64A) or deletion of the N-terminus (aa 1-64) results in reduced cell death activity of Bnip3. Moreover, mutation of a histidine residue in the C-terminal transmembrane domain to an alanine (Bnip3H173A) almost completely inhibits cell death activity of Bnip3. Bnip3C64A has reduced ability to interact with Bnip3, whereas Bnip3H173A is completely unable to interact with Bnip3, suggesting that homodimerization is important for Bnip3 function. A consequence of ischemia/reperfusion is the production of reactive oxygen species and oxidation of proteins, which promotes formation of disulfide bonds between proteins. Thus, these studies suggest that Bnip3 functions as a redox sensor where increased oxidative stress induces homodimerization and activation of Bnip3 via cooperation of the N-terminal cysteine residue and the C-terminal transmembrane domain.