αB-Crystallin (αBC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on Ser-19, 45, and 59 after stress. However, it is not known whether αBC phosphorylation directly affects cell survival. In the present study, constructs were prepared that encode forms of αBC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed αBC with Ser to Ala substitutions at all three positions, αBC(AAA), exhibited more stress-induced apoptosis. Cells expressing either αBC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing αBC(AAA), indicating that phosphorylation of Ser-59 confers protection. αBC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing αBC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing αBC(AAE). These results demonstrate that mimicking the phosphorylation of αBC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that αBC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of αBC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.