MicroRNAs are naturally existing, small, noncoding RNA molecules that downregulate posttranscriptional gene expression. Their expression pattern and function in the heart remain unknown. Here we report an array of microRNAs that are differentially and temporally regulated during cardiac hypertrophy. Significantly, the muscle-specific microRNA-1 (miR-1) was singularly downregulated as early as day 1 (0.56±0.036), persisting through day 7 (0.29±0.14), after aortic constriction–induced hypertrophy in a mouse model. Overexpression experiments showed that miR-1 inhibited its in silico–predicted, growth-related targets, including Ras GTPase–activating protein (RasGAP), cyclin-dependent kinase 9 (Cdk9), fibronectin, and Ras homolog enriched in brain (Rheb), in addition to protein synthesis and cell size. Thus, we propose that microRNAs play an essential regulatory role in the development of cardiac hypertrophy, wherein downregulation of miR-1 is necessary for the relief of growth-related target genes from its repressive influence and induction of hypertrophy.