MTF1 is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements (MREs). MTF1 responds to metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. We used multiple strategies to identify an unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, MTF1 expression increased, as did nuclear localization. Mtf1 knockdown impaired differentiation, while addition of non-toxic concentrations of Cu⁺ enhanced MTF1 expression and promoted myogenesis. Cu⁺ bound stoichiometrically to a C-terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes and binding was stimulated by copper. MTF1 formed a complex with MyoD at myogenic promoters, the master transcriptional regulator of the myogenic lineage. These studies establish novel mechanisms by which copper and MTF1 regulate gene expression in myoblast differentiation.