Background Micro RNA s (mi RNA s) play a key role in the development of heart failure, and recent studies have shown that the muscle‐specific miR‐1 is a key regulator of cardiac hypertrophy. We tested the hypothesis that chronic restoration of miR‐1 gene expression in vivo will regress hypertrophy and protect against adverse cardiac remodeling induced by pressure overload. Methods and Results Cardiac hypertrophy was induced by left ventricular pressure overload in male S prague‐ D awley rats subjected to ascending aortic stenosis. When the hypertrophy was established at 2 weeks after surgery, the animals were randomized to receive either an adeno‐associated virus expressing miR‐1 ( AAV 9. miR‐1 ) or green fluorescent protein ( GFP ) as control ( AAV 9. GFP ) via a single‐bolus tail‐vein injection. Administration of miR‐1 regressed cardiac hypertrophy (left ventricular posterior wall thickness,; 2.32±0.08 versus 2.75±0.07 mm, P <0.001) and (left ventricular septum wall thickness, 2.23±0.06 versus 2.54±0.10 mm, P <0.05) and halted the disease progression compared with control‐treated animals, as assessed by echocardiography (fractional shortening, 37.60±5.01% versus 70.68±2.93%, P <0.05) and hemodynamic analyses (end‐systolic pressure volume relationship/effective arterial elastance, 1.87±0.46 versus 0.96±0.38, P <0.05) after 7 weeks of treatment. Additionally, miR‐1 replacement therapy lead to a marked reduction of myocardial fibrosis, an improvement in calcium handling, inhibition of apoptosis, and inactivation of the mitogen‐activated protein kinase signaling pathways, suggesting a favorable effect on preventing the maladaptive ventricular remodeling. We also identified and validated a novel bona fide target of miR‐1 , Fibullin‐2 ( Fbln2 ), a secreted protein implicated in extracellular matrix remodeling. Conclusions Taken together, our findings suggest that restoration of miR‐1 gene expression is a potential novel therapeutic strategy to reverse pressure‐induced cardiac hypertrophy and prevent maladaptive cardiac remodeling.