The type 1 protein phosphatase (PP1) has been reported to be overactivated in the failing heart, leading to a depression in cardiac function. We investigated whether in vivo PP1 inhibition by myocardial gene transfer of inhibitor-2 (INH-2), an endogenous PP1 inhibitor, alleviates heart failure (HF) progression in the cardiomyopathic (CM) hamster, a well-established HF model. Adenoviral INH-2 gene delivery improved % fractional shortening of the left ventricle (LV) accompanied by reduced chamber size at 1 wk. In vivo myocardial INH-2 gene delivery induced an increase in cytosolic PP1 catalytic subunit alpha (PP1Calpha) without inducing the corresponding increase in cytosolic PP1 activity. On the other hand, INH-2 delivery induced a decrease in microsomal PP1Calpha, resulting in a preferential decrease in microsomal PP1 activity, thereby increasing in phospholamban phosphorylation at Ser16. INH-2 gene transfer alleviated brain natriuretic peptide expression, presumably reflecting improved cardiac function. Moreover, adeno-associated virus-mediated INH-2 gene delivery significantly extended the survival time for 3 mo. These results indicate that increased PP1 activity is an exacerbating factor during progression of genetic cardiomyopathy and modulation of PP1 activity by INH-2 provides a potential new treatment for HF without activating protein kinase A signaling in cardiomyocytes.