The cytosolic small heat shock protein, B-crystallin (BC), is a molecular chaperone expressed in large quantities in the heart, where it protects from stresses, such as ischemia/reperfusion (I/R). Upon I/R, p38 MAP kinase activation leads to the phosphorylation of BC on serine-59 (P-BC-S59), which increases its protective ability. BC confers protection, in part, by interacting with and affecting the functions of key components in stressed cells. In the present study, we investigated the hypothesis that protection from I/R damage in the heart by P-BC-S59 can be mediated by localizing to mitochondria. We found that P-BC-S59 localized with mitochondria isolated from untreated mouse hearts, and that this localization increased by more than 3-fold when the hearts were subjected to ex vivo I/R. Mitochondrial P-BC-S59 decreased when hearts were treated with the p38 inhibitor, SB202190. Moreover, compared to controls, SB202190 treated hearts exhibited increased tissue damage and reduced functional recovery upon reperfusion. I/R activates mitochondrial permeability transition pore opening, which increases cellular damage. We found that isolated mouse heart mitochondria incubated with a recombinant mutant form of BC that mimics P-BC-S59 exhibited decreased calcium-induced permeability transition pore opening, compared to mitochondria incubated with recombinant wild type BC. These results indicate that mitochondria may be among the key components in stressed cells with which P-BC-S59 interacts, and that this localization may protect the myocardium, in part, by modulating mitochondrial permeability transition pore opening, thus reducing I/R injury.