Myocyte enhancer factor 2 (MEF2) proteins play a key role in promoting the expression of muscle-specific genes in differentiated muscle cells. MEF2 activity is regulated by the association with several transcriptional co-factors and by post-translational modifications. In the present report, we provide evidence for a novel regulatory mechanism of MEF2C activity, which occurs at the onset of skeletal muscle differentiation and is based on Lys4 acetylation. This covalent modification results in the enhancement of MEF2C binding to DNA and chromatin. In particular, we report that the kinetic parameters of MEF2/DNA association change substantially upon induction of differentiation to give a more stable complex and that this effect is mediated by Lys4 acetylation. We also show that Lys4 acetylation plays a prominent role in the p300-dependent activation of MEF2C.