Immunoglobulin-like cell adhesion molecule (IgLON4) is a glycosylphosphatidylinositol-anchored membrane protein that has been associated with neuronal growth and connectivity, and its deficiency has been linked to increased fat mass and low muscle mass. Adequate information on IgLON4 is lacking, especially in the context of skeletal muscle. In this study, we report that IgLON4 is profusely expressed in mouse muscles and is intensely localized on the cell membrane. IgLON4 expression was elevated in CTX-injected mouse muscles, which confirmed its role during muscle regeneration, and was abundantly expressed at high concentrations at cell-to-cell adhesion and interaction sites during muscle differentiation. IgLON4 inhibition profoundly affected myotube alignment, and directional analysis confirmed this effect. Additionally, results demonstrating a link between IgLON4 and lipid rafts during myogenic differentiation suggest that IgLON4 promotes differentiation by increasing lipid raft accumulation. These findings support the notion that a well-aligned environment promotes myoblast differentiation. Collectively, IgLON4 plays a novel role in myogenesis and regeneration, facilitates myotube orientation, and is involved in lipid raft accumulation.