Enhancer of polycomb1 (Epc1) is an unsual member of the polycomb group gene family that regulates transcription. We previously reported that Epc1 regulated the skeletal muscle differentiation by recruiting homeobox gene (Hod), a transcriptional modulator. Here we propose that Epc1 might modulate skeletal muscle differentiation by interaction with serum response factor (SRF). Epc1-induced transactivation of 慣-skeletal actin was induced by co-transfection of SRF, a transcription factor regulating muscle-specific genes by binding DNA sequence known as CArG box. The result of promoter study using point mutation in 慣-skeletal actin suggested that CArG box was necessary to mediate synergistic interaction of Epc1 and SRF. By chromatin immunoprecipitation (ChIP) assay, it was observed that Epc1 binds to serum response element (SRE) in 慣-skeletal actin promoter in vivo. In gel shift assays performed with radiolabelled probes recognizing the CArG box of the 慣-skeletal actin promoter, Epc1 induced supershift of SRF/SRE complex, suggesting that Epc1 forms ternary complex with SRF. We further investigated possibility that Epc1 also involved in epigenetic regulation of 慣-skeletal actin expression. Immunoprecipitation analysis showed the physical interaction of Epc1 and p300, a histone acetyltransferases. Moreover Epc1 physically interacted with acetylated histones H3 and H4. In ChIP assays, Epc1 was shown to be involved in histone acetylation around 慣-skeletal actin promoter. These results suggest that Epc1 plays an important role in sarcomeric muscle differentiation by CArG box-dependent interaction with SRF and chromatin remodeling.