Cardiac hypertrophy involves a remodeling process of the heart in response to diverse pathological stimuli. Both calcineurin/nuclear factor of activated T cells pathway and microRNA-133 (miR-133) have been shown to play a critical role in cardiac hypertrophy. It has been recognized that the expression and activity of calcineurin increases and miR-133 expression decreases in the hypertrophic heart, and inhibition of calcineurin or increase of miR-133 expression protects against cardiac hypertrophy. Here we tested the interaction between miR-133 and calcineurin in cardiac hypertrophy. Cardiac hypertrophy in vivo and in vitro was induced by transverse aortic constriction and phenylephrine treatment. mRNA levels were measured by using real-time PCR methods. Luciferase assays showed that transfection of miR-133 in HEK293 cells downregulated calcineurin expression, which was reversed by cotransfection with the miR-133–specific 2′-O-methyl antisense inhibitory oligoribonucleotides. These results were confirmed in cultured primary cardiomyocytes. miR-133 expression was downregulated, and calcineurin activity was enhanced in both in vivo and in vitro cardiac hypertrophy models. Treatment of cells and animals with cyclosporin A, an inhibitor of calcineurin, prevented miR-133 downregulation. Moreover, the antisense oligodeoxynucleotides against the catalytic subunits of calcineurin Aβ and the decoy oligodeoxynucleotides targeting nuclear factor of activated T cells transcription factor, a calcineurin downstream effector, increased miR-133 expression in cultured primary cardiomyocytes. Our data show that reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy.