BACKGROUND: Cardiomyocyte hypertrophy is of central importance in the development of congestive heart failure. Whether proteins secreted from the myocardium itself contribute to myocardial hypertrophy is largely unknown. METHODS AND RESULTS: We performed a genetic yeast secretion trap screen using a murine cardiac cDNA library and identified 54 cardiac proteins that contained a secretion signal. When determining their mRNA expression in the myocardium of failing hearts, we found protease inhibitor 16 (PI16) to be strongly upregulated in hypertrophic and failing myocardium. PI16, a 489-amino acid protein with an unknown function, also displayed enhanced expression on the protein level after serum stimulation of primary cardiomyocytes and in failing myocardium. We found PI16 to be secreted rapidly by primary cardiomyocytes into the culture medium, where it inhibited cardiomyocyte growth. RNA interference-mediated suppression of endogenous PI16 in primary cardiomyocytes significantly enhanced cardiomyocyte size. Transgenic mice overexpressing PI16 in a cardiomyocyte-specific manner showed normal cardiac function but had smaller hearts with hypotrophic cardiomyocytes. CONCLUSIONS: Taken together, we identified 54 putatively secreted cardiac proteins. PI16, a novel protein secreted from the heart, is strongly upregulated early in heart failure and inhibits growth of cardiomyocytes both in vitro and in vivo. PI16 might represent a novel therapeutic target in heart failure.