Cardiac hypertrophy is maladaptive remodeling of the myocardium that often progresses to heart failure. The hypertrophic transport metabolon consists of membrane transporters implicated in hypertrophy, and substrates for these transporters are provided by the catalytic action of carbonic anhydrase II (CAII). CAII inhibition prevents and reverts cardiomyocyte hypertrophy in vitro, suggesting that CAII is an important protein in the development of hypertrophy. To explore this further, neonatal rat ventricular myocytes (NRVMs) were transduced with adenoviral constructs to overexpress wild type or catalytically inactive CAII. Upon treatment with phenylephrine, NRVMs overexpressing catalytically inactive CAII exhibited reduced cardiac remodeling associated with hypertrophy. Cardiomyocytes isolated from a line of mice with a null mutation in the CAII gene were also protected from hypertrophic stimulation. The findings of these studies support the importance of CAII in the promotion of cardiac hypertrophy, and may potentially lead to new therapeutic strategies for heart failure.