Cardiomyocyte hypertrophy is transcriptionally controlled and inhibited by glycogen synthase kinase 3β (GSK3β). Myocardin is a muscle-specific transcription factor with yet unknown relation to hypertrophy. Therefore, we investigated whether myocardin is sufficient to induce cardiomyocyte hypertrophy and whether myocardin is regulated by GSK3β through site-specific phosphorylation. Adenoviral myocardin overexpression induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes, with increased cell size, total protein amount, and transcription of atrial natriuretic factor (ANF). In vitro and in vivo (HEK 293 cells) kinase assays with synthetic peptides and full-length myocardin demonstrated that myocardin was a “primed” GSK3β substrate, with serines 455 to 467 and 624 to 636 being the major GSK3β phosphorylation sites. Myocardin-induced ANF transcription and increase in total protein amount were enhanced by GSK3β blockade (10 mmol/L LiCl), indicating that GSK3β inhibits myocardin. A GSK3β phosphorylation-resistant myocardin mutant (8xA) activated ANF transcription twice as potently as wildtype myocardin under basal conditions with GSK3β being active. Conversely, a GSK3β phospho-mimetic myocardin mutant (8xD) was transcriptionally repressed after GSK3β blockade, indicating that GSK3β phosphorylation at the sites identified inhibits myocardin transcriptional activity. GAL4-myocardin fusion constructs demonstrated that GSK3β phosphorylation reduced the intrinsic myocardin transcriptional activity. A cell-permeable (Antennapedia protein transduction tag) peptide containing the mapped myocardin GSK3β motifs 624 to 636 induced hypertrophy of cultured cardiomyocytes, suggesting that the peptide acted as substrate-based GSK3β inhibitor in cardiomyocytes. Therefore, we conclude that the GSK3β–myocardin interaction constitutes a novel molecular control of cardiomyocyte hypertrophy. Phosphorylation by GSK3β comprises a novel post-transcriptional regulatory mechanism of myocardin.