NEW FINDINGS: What is the central question of this study? How are the dynamics of IL-15 and its receptors altered during the differentiation of myoblasts into myotubes, and how is IL-15 regulated? What is the main finding and its importance? ABSTRACT: Interleukin-15 (IL-15) is a myokine in the Interleukin-2 (IL-2) family that is generated in the skeletal muscle during exercise. The functional effect of IL-15 involves muscle regeneration and metabolic regulation in skeletal muscle. Reports have indicated that the mechanism of Interleukin-15 receptor subunit alpha (IL-15RA) regulates IL-15 localization in immune cells. However, the dynamic of IL-15 and its receptors, which regulate the IL-15 pathway in skeletal muscle differentiation, have not yet been clarified. This study investigated the mechanism of IL-15 regulation using a mouse skeletal muscle cell line, C2C12 cells. We found that the mRNA expression of IL-15, Interleukin 2 Receptor Subunit Beta (IL-2RB) (CD122), and Interleukin 2 Receptor Subunit Gamma (IL-2RG) (CD132) increased, but that IL-15RA exhibits different kinetics as differentiation progresses. We also found that IL-15, mainly localized in the cytosol, preassembled with IL-15RA in the cytosol and fused to the plasma membrane. Moreover, IL-15RA increased IL-15 protein levels. Our findings suggest that genes comprising the IL-15 signaling complex are enhanced with the differentiation of myotubes and that IL-15RA regulates the protein kinetics of IL-15 signaling in skeletal muscle. This article is protected by copyright. All rights reserved.