Recombinant adeno-associated virus (rAAV)-based gene therapy represents a promising approach for the treatment of heart muscle diseases, but the molecular mechanisms that direct rAAV transduction remain unclear. Here we demonstrate that ?-adrenergic receptor stimulation with isoproterenol (ISO) markedly increased cardiomyocyte transduction of rAAV in vitro and in vivo. Conversely, chronic ?-adrenergic receptor downregulation significantly suppressed rAAV transduction. Pretreatment with calcium signaling cascade inhibitors including calcineurin inhibitory peptide (CNIP) strongly suppressed the positive effects of ISO on rAAV transduction. Additionally we document that ISO treatment led to a significant increase in double-stranded (ds) DNA synthesis of the rAAV genome and an increase in promoter activity. Moreover, stimulation with ISO did not affect rAAV transduction in calcineurin nullizygous mice. Collectively, we conclude that a calcium-dependent pathway regulates rAAV vector transduction at a number of stages that may include vector mobilization, conversion, and transcription activity. Modulating this pathway through ?-adrenergic signaling enhances rAAV-mediated gene delivery to cardiomyocytes, and may be valuable when considering therapeutic approaches for heart muscle diseases.