Elevated phosphorylation of AMP-activated kinase (AMPK) has been shown to inhibit skeletal muscle growth in both culture and animal models, but its role in differentiation of muscle cells is less clear. P21 is a known to have an important role in differentiation, but AMPK's role regulating p21 in differentiation in muscle cultures is unknown. Therefore, the purpose of this study was to determine the role of p21 in differentiation of skeletal muscle cells under conditions of elevated AMPK phosphorylation. Treating C2C12 myoblast cultures with 1mM 5-aminoimidazole-4-carboxamide 1--D-ribonucleoside (AICAR) for up to 24 hr induced AMPK phosphorylation. Activation of AMPK reduced p21 protein and mRNA expression, which was associated with reduced G1-S cell cycle transition and p21 promoter activity. AICAR treated myoblasts undergoing differentiation also had reduced p21 protein expression, reduced myotube formation, and myosin accumulation. When myotube cultures were treated with AICAR for 24hr, p21, myosin protein expression, and MyoD were significantly reduced. Myotube atrophy was also apparent when compared to control conditions. Addition of Compound C, an AMPK inhibitor attenuated AICARs negative effects on the myotube cultures. The nuclear expression of p21 protein appeared to be more affected by AICAR treated myotubes than the cytosolic portion of p21 protein, which was attenuated with Compound C treatment. Further analysis revealed that AICAR treatment increased PGC-1 and decreased FOXO3A protein expression, which was reversed with Compound C co-treatment. Knockdown of PGC-1 with shRNA corroborated the Compound C data, preserving nuclear FOXO3A and p21 protein expression. These data demonstrate that AICAR-induced AMPK phosphorylation inhibits cell cycle transition, reducing differentiation of myoblasts into myotubes, through PGC-1-FOXO3A-p21.