The nuclear factor of activated T-cells (NFAT) c1 has been shown to be essential for Ca²⁺-dependent upregulation of myosin heavy chain (MyHC) I/ß expression during skeletal muscle fiber type transformation. To investigate the molecular mechanism of the regulation of MyHCI/ß gene expression, several techniques like transient transfections, luciferase reporter gene assays, RNA interference assays, Western blot analysis, RT-PCR, in vitro kinase assays, immunofluorescence studies, and electrophoretic mobility shift assays were used. Here, we report activation of extracellular signal-regulated kinase (ERK) 1/2 in Ca²⁺-ionophore-treated C2C12 myotubes and electrostimulated mouse soleus muscle. Activated ERK1/2 enhanced NFATc1-dependent upregulation of a –2.4 kb MyHCI/ß promoter construct without affecting subcellular localisation of endogenous NFATc1. Instead, ERK1/2 augmented phosphorylation of transcriptional coactivator p300, promoted its recruitment to NFATc1 and increased NFATc1-DNA binding to a –439/–432 bp NFAT site of the MyHCI/ß promoter. In line, inhibition of ERK1/2 signaling abolished the effects of p300. Comparison between wild-type p300 and an acetyltransferase-deficient mutant (p300DY) indicated increased NFATc1-DNA binding as a consequence of p300-mediated acetylation of NFATc1. Activation of the MyHCI/ß promoter by p300 depends on two conserved acetylation sites in NFATc1, which affect DNA binding and transcriptional stimulation. NFATc1 acetylation occurred in Ca²⁺-ionophore treated C2C12 myotubes and electrostimulated mouse soleus. Finally, endogenous MyHCI/ß gene expression in C2C12 myotubes was strongly inhibited by p300DY and a mutant deficient in ERK phosphorylation sites. In conclusion, ERK1/2-mediated phosphorylation of p300 is crucial for enhancing NFATc1 transactivation function by acetylation, which is essential for Ca²⁺-induced MyHCI/ß expression.