Background: Myopathy affects nearly half of individuals with alcohol use disorder (AUD), and impaired skeletal muscle regenerative potential is a probable contributing factor. Previous findings from our laboratory indicate that chronic in vivo and in vitro ethanol (EtOH) treatment decreases myogenic potential of skeletal muscle myoblasts. Myogenesis, a highly coordinated process, requires shifts in cellular metabolic state allowing for myoblasts to proliferate and differentiate into mature myotubes. The objective of this study was to determine whether alcohol interferes with myoblast mitochondrial and glycolytic metabolism and impairs myogenic differentiation. Methods: Myoblasts were isolated from vastus lateralis muscle excised from alcohol-na€ ıve adult male (n = 5) and female (n = 5) rhesus macaques. Myoblasts were proliferated for 3 days (day 0 differentiation; D0) and differentiated for 5 days (D5) with or without 50 mM EtOH. Metabolism was assessed using a mitochondrial stress test to measure oxygen consumption (OCR) and extracellular acidification (ECAR) rates at D0. Differentiation was examined at D5. Expression of mitochondrial and glycolytic genes and mitochondrial DNA (mtDNA) was measured at D0 and D5. Results: Ethanol significantly (p < 0.05) increased myoblast maximal OCR and decreased ECAR at D0, and decreased fusion index, myotubes per field, and total nuclei at D5. The EtOH-induced decrease in ECAR was associated with the EtOH-mediated decreases in fusion index and myotubes per field. EtOH did not alter the decrease in glycolytic gene expression and increase in mtDNA from D0 to D5. Conclusion: During myoblast proliferation, EtOH decreased glycolytic metabolism and increased maximal OCR, suggesting that myoblast metabolic phenotype was dysregulated with EtOH. The EtOH-induced decrease in ECAR was associated with decreased differentiation. These findings suggest that EtOH-mediated shifts in metabolic phenotype may underlie impaired differentiation, which has important clinical implications for myogenesis in those affected by alcoholic myopathy.