Myostatin is well established as a negative regulator of skeletal muscle growth, but its role in the heart is controversial. Our goal in this study was to characterize myostatin regulation following cardiomyocyte stress and to examine the role of myostatin in the regulation of cardiomyocyte size. Neonatal cardiomyocytes were cultured and stressed with phenylephrine. Adenovirus was used to overexpress myostatin or dominant negative myostatin in culture. Adeno-associated virus was used to overexpress myostatin or dominant negative myostatin in mice. Myostatin is upregulated following cardiomyocyte stress in an Erk-dependent manner that is associated with increased nuclear translocation and DNA binding activity of MEF-2. Myostatin overexpression leads to decreased and myostatin inhibition to increased cardiac growth both in vitro and in vivo due to modulation of Akt and NFAT3 pathways. Myostatin is a negative regulator of cardiac growth, and further studies are warranted to investigate the role of myostatin in the healthy and failing heart.