Skeletal muscle stem cells play a critical role in regeneration of myofibers. We have previously demonstrated that chronic binge alcohol (CBA) markedly attenuates myoblast differentiation potential and myogenic gene expression. Muscle specific microRNAs are implicated in regulating myogenic genes. The aim of this study was to determine whether myoblasts isolated from asymptomatic CBA administered SIV-infected macaques treated with antiretroviral therapy (ART) showed similar impairments and if so, to elucidate potential underlying mechanisms. Myoblasts were isolated from muscle at 11 months post-SIV infection from CBA/SIV and from time matched sucrose-treated SIV-infected (SUC/SIV) macaques and controls. Myoblasts isolated from SUC/SIV and CBA/SIV animals had a significant decrease in myoblast differentiation and myogenic gene expression compared to myoblasts from controls. SIV and CBA decreased muscle specific, miR-206, in plasma and muscle and SIV decreased miR-206 expression in myoblasts, with no statistically significant changes in other muscle specific microRNAs. This was associated with an observed significant increase in histone deacetylase 4 (HDAC4) and decrease in myogenic enhancer factor 2C (MEF2C) expression in CBA/SIV muscle. Transfection with miR-206 inhibitor decreased myotube differentiation, increased expression of HDAC4, and decreased MEF2C, suggesting a critical role of miR-206 in myogenesis. Moreover, HDAC4 was confirmed as a direct miR-206 target. These results support a mechanistic role for decreased miR-206 in suppression of myoblast differentiation resulting from chronic alcohol and SIV infection. The parallel changes in skeletal muscle and circulating levels of miR-206 warrant studies to establish the possible use of plasma miR-206 as an indicator of impaired muscle function.