Myoblast fusion is a highly regulated process that is important during muscle development and myofiber repair and is also likely to play a key role in the incorporation of donor cells in myofibers for cell-based therapy. Although several proteins involved in muscle cell fusion in Drosophila are known, less information is available on the regulation of this process in vertebrates, including humans. To identify proteins that are regulated during fusion of human myoblasts, microarray studies were performed on samples obtained from human fetal skeletal muscle of seven individuals. Primary muscle cells were isolated, expanded, induced to fuse in vitro, and gene expression comparisons were performed between myoblasts and early or late myotubes. Among the regulated genes, melanoma cell adhesion molecule (M-CAM) was found to be significantly downregulated during human fetal muscle cell fusion. M-CAM expression was confirmed on activated myoblasts, both in vitro and in vivo, and on myoendothelial cells (M-CAM(+) CD31(+)), which were positive for the myogenic markers desmin and MyoD. Lastly, in vitro functional studies using M-CAM RNA knockdown demonstrated that inhibition of M-CAM expression enhances myoblast fusion. These studies identify M-CAM as a novel marker for myogenic progenitors in human fetal muscle and confirm that downregulation of this protein promotes myoblast fusion.