The complex process of muscle differentiation is organized predominantly by the myogenic regulatory factors, Myf5, MyoD, Myf6 and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. The regulation of myogenin at the transcriptional level is well documented and has been studied in depth to reveal a multilayer, robust regulation of its expression. The possibility that myogenin could be regulated at the post transcriptional level through microRNAs has yet to be identified. MicroRNAs are non-coding RNA molecules that post-transcriptionally regulate many cellular processes. In an effort to investigate the role microRNAs play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491 , miR-329 and miR-96) were predicted to bind the myogenin 3 'untranslated region. However, luciferase assays showed only miR-186 inhibited translation and 3 'untranslated region mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during muscle cell differentiation. Functional studies demonstrated that cellular levels of miR-186 were inversely proportional to those of myogenin throughout differentiation and that its overexpression inhibited the differentiation of C2C12 and primary muscle cells. Alternatively, inhibition of miR-186 during muscle differentiation increased the capacity of muscle cells to differentiate raising the possibility that a miR- 186 inhibitor could be utilized in muscle damage therapeutics. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation, providing an additional layer of regulation to the robust regulation of myogenin expression.