MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate gene expression. Although miRNAs are highly expressed in the heart, their roles in heart diseases are currently unclear. Using microarray analysis designed to detect the majority of mammalian miRNAs identified thus far, we demonstrated that miRNAs are aberrantly expressed in hypertrophic mouse hearts. The time course of the aberrant miRNA expression was further identified in mouse hearts at 7, 14, and 21 days after aortic banding. Nineteen of the most significantly dysregulated miRNAs were further confirmed by Northern blot and/or real-time polymerase chain reaction, in which miR-21 was striking because of its more than fourfold increase when compared with the sham surgical group. Similar aberrant expression of the most up-regulated miRNA, miR-21, was also found in cultured neonatal hypertrophic cardiomyocytes stimulated by angiotensin II or phenylephrine. Modulating miR-21 expression via antisense-mediated depletion (knockdown) had a significant negative effect on cardiomyocyte hypertrophy. The results suggest that miRNAs are involved in cardiac hypertrophy formation. miRNAs might be a new therapeutic target for cardiovascular diseases involving cardiac hypertrophy such as hypertension, ischemic heart disease, valvular diseases, and endocrine disorders.