The ubiquitin-proteasome pathway is essential for skeletal muscle growth and development. Proteasomes generate oligopeptides in the cytoplasm, and these peptides are considered to be rapidly degraded to amino acids by several intracellular aminopeptidases. However, the role of intracellular aminopeptidases in muscle growth remains unknown. In this study, therefore, we investigated the role of intracellular aminopeptidases in C2C12 myoblast proliferation and differentiation. Inhibition of intracellular aminopeptidases by Bestatin methyl ester (Bes-ME) decreased leucine and alanine aminopeptidase activity, and impaired proliferation and differentiation of C2C12 myoblasts. Furthermore, we observed that the inhibition of intracellular aminopeptidases reduced intracellular levels of amino acid and ATP level, and suppressed the phosphorylation of the mTOR pathway. These results suggested that intracellular aminopeptidases affect C2C12 myoblast proliferation and differentiation via mTOR pathway; however, further studies are required to clarify the role of aminopeptidase in skeletal muscle. (C) 2020 The Authors. Published by Elsevier Inc.