The mitochondrial network in muscle is controlled by the opposing processes of mitochondrial biogenesis and mitophagy. The coactivator PGC-1α regulates biogenesis, while the transcription of mitophagy-related genes is controlled by transcription factor EB (TFEB). PGC-1α activation is induced with exercise, however the effect of exercise on TFEB is not fully known. We investigated the interplay between PGC-1α and TFEB on mitochondria in response to acute contractile activity in C2C12 myotubes, and following exercise in WT and PGC-1α KO mice. TFEB nuclear localization was increased by 1.6-fold following 2 hours of acute myotube contractile activity. TFEB transcription and LC3 localization to mitochondria were also simultaneously increased by 2-3-fold. Viral overexpression of TFEB increased PGC-1α and COXIV gene expression. In WT mice, TFEB translocation to the nucleus increased 2.4-fold in response to acute exercise, while TFEB transcription, assessed through the electroporation of a TFEB promoter construct, was elevated by 4-fold. These exercise effects were dependent on the presence of PGC-1α. Our data suggest that acute exercise provokes TFEB expression and activation both in vitro and in vivo, in a PGC-1α-dependent manner. Our results indicate that TFEB, along with PGC-1α, are important regulators of mitochondrial biogenesis in muscle as a result of exercise.