The purpose of the present study was to investigate the effect of interferon (IFN)-γ on the transcriptomic profile of differentiating mouse C2C12 myogenic cells. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through real-time PCR. IFN-γ (1 ng/mL) increased myoblast proliferation but decreased cell respiration and myosin heavy chain content and slightly decreased the fusion index in differentiating C2C12 cell cultures. The genes upregulated through IFN-γ were involved in cell cycle; regulation of cell proliferation; programmed cell death; chemotaxis; and cytokine, growth factor, and peptidase activity, whereas the genes downregulated through IFN-γ primarily contributed to the regulation of transcription, cell-cell signaling, nitrogen compound biosynthesis, ser/thr protein kinase signaling, and regulation of the Wnt pathway. In conclusion, IFN-γ affects the expression of numerous genes associated with the regulation of several processes in myogenesis. The effects of IFN-γ on cellular transcription include (1) alteration of cytokine/growth factor expression, promoting cell proliferation and migration but inhibiting differentiation, (2) impairment of pro-myogenic transcription, (3) disruption of cell adhesion and sarcolemma/cytoskeleton organization, and (4) increased peptidase activity leading to enhanced proteolysis and apoptosis.