The induction of Rb gene expression is a key event in the process of myogenic differentiation. We recently demonstrated that transcription of the Rb gene is repressed by MIZF, previously characterized as an MBD2-binding partner. Here we show the new roles of MIZF as a regulator of myogenic differentiation. MIZF mRNA was detected in undifferentiated C2C12 myoblasts but its expression decreased during myogenesis, correlating with an increase in Rb mRNA. To examine the function of MIZF in regulating myogenic differentiation, we transduced C2C12 myoblasts with adenoviral vectors to constitutively produce MIZF at high levels. When switched to differentiation medium, these cells showed decreased expression of Rb as well as differentiation markers such as myogenin and Troponin-T, and consequently could not differentiate into multinucleated myotubes. These results suggest that transcriptional repression of Rb by MIZF could be one of the critical determinants in myogenic differentiation.