Myocardin, a serum response factor (SRF)-dependent cofactor, is a potent activator of smooth muscle gene activity but a poor activator of cardiogenic genes in pluripotent 10T1/2 fibroblasts. Posttranslational modification of GATA4, another myocardin cofactor, by sumoylation strongly activated cardiogenic gene activity. Here, we found that myocardin's activity was strongly enhanced by SUMO-1 via modification of a lysine residue primarily located at position 445 and that the conversion of this residue to arginine (K445R) impaired myocardin transactivation. PIAS1 was involved in governing myocardin activity via its E3 ligase activity that stimulated myocardin sumoylation on an atypical sumoylation site(s) and by its physical association with myocardin. Myocardin initiated the expression of cardiac muscle-specified genes, such as those encoding cardiac alpha-actin and alpha-myosin heavy chain, in an SRF-dependent manner in 10T1/2 fibroblasts, but only in the presence of coexpressed SUMO-1/PIAS1. Thus, SUMO modification acted as a molecular switch to promote myocardin's role in cardiogenic gene expression.