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Exercise is an important strategy in the prevention and treatment of metabolic diseases, like diabetes and obesity. Alterations in the skeletal muscle proteome, including post-translational modifications, especially acetylation, regulate its metabolic adaptations to exercise. Here, we examined the effect of 6-week aerobic exercise and Scriptaid, a HDAC4/5 inhibitor, on the proteome and acetylome of skeletal muscle in mice. We find Scriptaid and exercise both induce acetylation modification changes of some proteins involved in metabolism, suggest that exercise improves metabolic health by regulating protein acetylation level.