Both protein kinase Cα-dependent Na+/Ca2+ exchanger1 (NCX1) phosphorylation and calcineurin activity are required for the depression of NCX activity observed in chronically phenylephrine (PE)-treated hypertrophic neonatal rat cardiomyocytes. In this study, we explored the possibility that the same changes occur in vivo hypertrophy. In the hypertrophic hearts of thoracic aortic-banded (TAB) mice, NCX1 phosphorylation increased significantly compared with control hearts. Furthermore, the TAB-induced cardiac hypertrophy was much less prominent in transgenic mice overexpressing an NCX1 mutant having defective phosphorylation sites. These data suggest that the phosphorylation status of NCX1 may play an important role in the pathogenesis of load-induced cardiac hypertrophy.