Calcineurin, which binds to the Z-disc in cardiomyocytes via α-actinin, promotes cardiac hypertrophy in response to numerous pathologic stimuli. However, the endogenous mechanisms regulating calcineurin activity in cardiac muscle are not well understood. We demonstrate that a muscle-specific F-box protein called atrogin-1, or muscle atrophy F-box, directly interacts with calcineurin A and α-actinin-2 at the Z-disc of cardiomyocytes. Atrogin-1 associates with Skp1, Cul1, and Roc1 to assemble an SCF atrogin-1 complex with ubiquitin ligase activity. Expression of atrogin-1 decreases levels of calcineurin A and promotes its ubiquitination. Moreover, atrogin-1 attenuates agonist-induced calcineurin activity and represses calcineurin-dependent transactivation and NFATc4 translocation. Conversely, downregulation of atrogin-1 using adenoviral small interfering RNA (siRNA) expression enhances agonist-induced calcineurin activity and cardiomyocyte hypertrophy. Consistent with these cellular observations, overexpression of atrogin-1 in hearts of transgenic mice reduces calcineurin protein levels and blunts cardiac hypertrophy after banding of the thoracic aorta. These studies indicate that the SCF atrogin-1 ubiquitin ligase complex interacts with and represses calcineurin by targeting calcineurin for ubiquitin-mediated proteolysis, leading to inhibition of cardiac hypertrophy in response to pathologic stimuli.