Background: Skeletal myogenesis is orchestrated by the precise regulation of multiple transcription factors. Previously, we demonstrated that enhancer of polycomb 1 (Epc1) interacts with serum response factor (SRF) to induce skeletal muscle differentiation. Here we report that an interacting partner of Epc1, ret finger protein (RFP, also known as TRIM27), negatively regulates muscle differentiation. Methods & Results: By yeast two-hybrid screening and immunoprecipitation, RFP was shown to interact with Epc1. RFP was highly expressed in adult skeletal muscles and was down-regulated during myoblast differentiation. RFP overexpression delayed myogenic differentiation. Epc1-induced enhancements of SRF-dependent multinucleation, transactivation of the skeletal 慣-actin promoter, and muscle-specific gene induction were abolished by cotransfection of RFP. Immunoprecipitation analysis revealed that transfection of RFP interfered with the interaction between Epc1 and SRF. In the presence of Epc1, RFP reduced binding of SRF to the serum response element (SRE) of the skeletal 慣-actin promoter. RFP also reduced the binding of Epc1 to the SRE. The expression of myogenin, MyoD, and muscle creatine kinase was upregulated in hamstring muscles obtained from Rfp-/- mice. Isolated myoblasts from Rfp knockout mice showed enhanced myoblast differentiation. Conclusions:Taken together, our findings establish the function of RFP as a novel transcriptional modulator that inhibits Epc1/SRF-induced muscle differentiation.