AbstractDifferentiation of skeletal muscle myoblasts involves activation of muscle‐specific markers such as MyoD, Myf5, MRF4, and myogenin, followed by exit from the cell cycle, expression of structural proteins, and fusion into multinucleated myotubes. Cyclin D3 is upregulated during muscle differentiation, and expression of cyclin D3 in proliferating myoblasts causes early activation of myogenesis. In this study, we have identified the genes activated by cyclin D3 expression in C2C12 myoblasts and differentiated cells by real‐time PCR analysis. Cyclin D3 expression induced faster differentiation kinetics and increase in levels of myogenic genes such as MyoD, Myf5, and myogenin at an early stage during the differentiation process, although long‐term myogenic differentiation was not affected. Transcript levels of the transcription factor Pax7 that is expressed in muscle progenitors were enhanced by cyclin D3 expression in myoblasts. Components of a histone methyltransferase complex recruited by Pax7 to myogenic gene promoters were also regulated by cyclin D3. Further, the Pax7 promoter was upregulated in myoblasts expressing cyclin D3. Myoblasts that expressed cyclin D3 showed moderately higher levels of the cyclin‐dependent kinase inhibitor p21 and were stalled in G2/M phase of the cell cycle. Our findings suggest that cyclin D3 primes myoblasts for differentiation by enhancing muscle specific gene expression and cell cycle exit. J. Cell. Biochem. 113: 209–219, 2012. © 2011 Wiley Periodicals, Inc.