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CCL5/RANTES was identified as a novel exercise-reducible myokine.Ca2+- and AMPK-dependent signaling participates in the CCL5 reduction.Voluntary exercise reduced serum CCL5 levels in mice.Voluntary exercise reduced Ccl5 expression in fast-twitch skeletal muscles in mice.Skeletal muscle is an endocrine organ that secretes several proteins, which are collectively termed myokines. Although many studies suggest that exercise regulates myokine secretion, the underlying mechanisms remain unclear and all the exercise-dependent myokines have not yet been identified. Therefore, in this study, we attempted to identify novel exercise-dependent myokines by using our recently developed in vitro contractile model. Differentiated C2C12 myotubes were cultured with or without electrical pulse stimulation (EPS) for 24 h to induce cell contraction, and the myokines secreted in conditioned medium were analyzed using a cytokine array. Although most myokine secretions were not affected by EPS, the secretion of Chemokine (C-C motif) ligand 5 (CCL5) (regulated on activation, normal T cell expressed and secreted (RANTES)) was significantly reduced by EPS. This was further confirmed by ELISA and quantitative PCR. Contraction-dependent calcium transients and activation of 5′-AMP activating protein kinase (AMPK) appears to be involved in this decrease, as the chelating Ca2+ by EGTA blocked contraction-dependent CCL5 reduction, whereas the pharmacological activation of AMPK significantly reduced it. However, Ccl5 gene expression was increased by AMPK activation, suggesting that AMPK-dependent CCL5 decrease occurred via post-transcriptional regulation. Finally, mouse experiments revealed that voluntary wheel-running exercise reduced serum CCL5 levels and Ccl5 gene expression in the fast-twitch muscles. Overall, our study provides the first evidence of an exercise-reducible myokine, CCL5, in the mouse skeletal muscle. Although further studies are required to understand the precise roles of the skeletal muscle cell contraction-induced decrease in CCL5, this decrease may explain some exercise-dependent physiological changes such as those in immune responses.