Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here, we show that αB-crystallin confers protection to FAK against calpain-mediated proteolysis under mechanical stress in cardiomyocytes. Biochemical assays, chemical cross-linking coupled to mass spectrometry experiments, mutational analyses and Förster resonance energy transfer (FRET) were combined to investigate the basis of FAK and αB-crystallin interaction. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation by mechanical stress. αB-crystallin silencing results in calpain-dependent FAK depletion and in increased apoptosis of cardiomyocytes. The overexpression of a myc-FAK construct or treatment with a calpain inhibitor (E64) restored the survival of cardiomyocytes depleted of αB-crystallin. The association between FAK and αB-crystallin was also demonstrated to occur in response to pressure overload in rat left ventricle. The myocardial depletion of αB-crystallin by siRNA results in enhanced apoptosis of cardiomyocytes and myocardial fibrosis in overloaded hearts. These alterations were markedly attenuated in the overloaded left ventricles of transgenic mice with cardiac specific FAK expression. These findings define a novel mechanism by which αB-crystallin controls FAK function, with impact on cardiomyocyte survival and cardiac remodelling in response to stress.