Activation of protein kinase A (PKA) by elevation of the intracellular cyclic AMP (cAMP) level inhibits skeletal myogenesis. Previously, an indirect modulation of the myogenic regulatory factors (MRFs) was implicated as the mechanism. Because myocyte enhancer factor 2 (MEF2) proteins are key regulators of myogenesis and obligatory partners for the MRFs, here we assessed whether these proteins could be involved in PKA-mediated myogenic repression. Initially, in silico analysis revealed several consensus PKA phosphoacceptor sites on MEF2, and subsequent analysis by in vitro kinase assays indicated that PKA directly and efficiently phosphorylates MEF2D. Using mass spectrometric determination of phosphorylated residues, we document that MEF2D serine 121 and serine 190 are targeted by PKA. Transcriptional reporter gene assays to assess MEF2D function revealed that PKA potently represses the transactivation properties of MEF2D. Furthermore, engineered mutation of MEF2D PKA phosphoacceptor sites (serines 121 and 190 to alanine) rendered a PKA-resistant MEF2D protein, which efficiently rescues myogenesis from PKA-mediated repression. Concomitantly, increased intracellular cAMP-mediated PKA activation also resulted in an enhanced nuclear accumulation of histone deacetylase 4 (HDAC4) and a subsequent increase in the MEF2D-HDAC4 repressor complex. Collectively, these data identify MEF2D as a primary target of PKA signaling in myoblasts that leads to inhibition of the skeletal muscle differentiation program.