Hypoxic or ischemic apoptosis is often tightly associated with the opening of mitochondrial permeability transition pore (mPTP); however, the molecular mechanisms regulating mPTP and thus mitochondrial-dependent apoptosis remain elusive. Emerging evidence indicates that the movement of key proteins in or out of mitochondria play a critical regulatory role in apoptosis. Here, we reported that, unexpectedly, the microtubule-associated protein 4 (MAP4) translocated from cytosol to mitochondria upon phosphorylation after hypoxia treatment in neonatal cardiomyocytes. When targeted to mitochondria, MAP4 was found to lead to mPTP opening and induce apoptosis. Mitochondrial accumulation and pro-apoptotic function of MAP4 could be reversed through the genetic inhibition of MAP4 phosphorylation. The MAP4(Ala) mutant, which mimicked the dephosphorylated form, suppressed mitochondrial translocation and apoptosis. Our data reveal a novel role of MAP4 in cardiac apoptosis and suggest a potential therapeutic strategy targeting mitochondrial translocation of MAP4 against apoptotic heart diseases.