Objective: Strategies improving skeletal muscle mitochondrial capacity are commonly paralleled by improvements in (metabolic) health. We and others previously identified microRNAs regulating mitochondrial oxidative capacity, but data in skeletal muscle are limited. Therefore, the present study aimed to identify novel microRNAs regulating skeletal muscle mitochondria! metabolism. Methods and results: We conducted an unbiased, hypothesis-free microRNA silencing screen in C2C12 myoblasts, using >700 specific microRNA inhibitors, and investigated a broad panel of mitochondria! markers. After subsequent validation in differentiated C2C12 myotubes, and exclusion of microRNAs without a human homologue or with an adverse effect on mitochondria] metabolism, 19 candidate microRNAs remained. Human clinical relevance of these microRNAs was investigated by measuring their expression in human skeletal muscle of subject groups displaying large variation in skeletal muscle mitochondrial capacity. Conclusion: The results show that that microRNA-320a, microRNA-196b-3p, microRNA-150-5p, and microRNA-34c-3p are tightly related to in vivo skeletal muscle mitochondrial function in humans and identify these microRNAs as targets for improving mitochondrial metabolism. (C) 2017 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license.