Exosomes are vesicles released by many eukaryotic cells; their cargo includes proteins, mRNA and microRNA (miR) that can be transferred to recipient cells and regulate cellular processes in an autocrine or paracrine manner. While cells of the myoblast lineage secrete exosomes, it is not known whether skeletal muscle fibers (myofibers) release exosomes. In this study, we found that cultured myofibers release nanovesicles that have bilamellar membranes and an average size of 60-130 nm, contain typical exosomal proteins and miRNAs and are taken up by C2C12 cells. miR-133a was found to be the most abundant myomiR in these vesicles while miR-720 was most enriched in exosomes compared to parent myofibers. Treatment of NIH 3T3 cells with myofiber-derived exosomes downregulated the miR-133a targets proteins Smarcd1 and Runx2, confirming that these exosomes have biologically relevant effects on recipient cells. Denervation resulted in a marked increase in miR-206 and reduced expression of miRs 1, 133a, and 133b in myofiber-derived exosomes. These findings demonstrate that skeletal muscle fibers release exosomes which can exert biologically significant effects on recipient cells, and that pathological muscle conditions such as denervation induce alterations in exosomal miR profile which could influence responses to disease states through autocrine or paracrine mechanisms.