Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Cardiomyocyte hypertrophy is characterized by increased cell size and altered gene expression. By differential-display polymerase chain reaction and Western blotting we found that the transcriptional coactivator MBF1 was upregulated during hypertrophy in cardiomyocyte cultures. Furthermore, MBF1 protein level increased in two animal models of hypertrophy, angiotensin II treatment and aortic banding. MBF1 antisense oligodeoxynuclotides blocked phenylephrine-induced hypertrophy, suggesting MBF1 plays a key role in hypertrophic growth. In contrast, overexpression of MBF1 potentiated the hormone-induced response of the atrial natriuretic peptide promoter. MBF1 overexpressed by transient transfection cooperated with the transcription factor c-Jun in activation of transcription but not with GATA4. MBF1 and c-Jun induced the activity of a transiently transfected atrial natriuretic peptide promoter, whereas neither MBF1 nor c-Jun could induce the promoter alone. Moreover, MBF1 bound to c-Jun in vitro. These data suggest that MBF1 is a transcriptional coactivator of c-Jun regulating hypertrophic gene expression. Inhibitor studies suggested that MBF1 activates the atrial natriuretic peptide promoter independently of the calcineurin and CaMK signaling pathways. Our results indicate that MBF1 participates in hormone-induced cardiomyocyte hypertrophy and activates hypertrophic gene expression as a coactivator of c-Jun.