KLF10 is a member of the Krüppel-like family of transcription factors that is known to play important roles in musculoskeletal system homeostasis. KLF10 is highly expressed in skeletal muscle, yet little is known regarding its functions in this tissue. siRNA-mediated suppression of KLF10 in C2C12 myocyte precursor cells resulted in inhibition of myocyte differentiation and fusion as well as decreased mitochondrial numbers and ATP levels. RNAseq analysis identified 504 and 612 genes to be mis-regulated following knockdown of KLF10 in undifferentiated and differentiated C2C12 cells respectively. Differentially expressed genes were associated with impaired MyoD signaling, calcium signaling, skeletal muscle contraction and skeletal muscle function pathways. Mass-spectrometry based metabolomics analyses revealed alterations in a number of critical metabolites involved in the TCA cycle including lactate, succinate, aspartate and isocitrate following suppression of KLF10 potentially explaining some of the defects observed with regard to ATP production in KFL10 depleted cells. To translate these findings in vivo, we assessed the impact of KLF10 knockout on slow and fast twitch muscles of mice. Electron microscopic analyses revealed striking defects in skeletal muscle architecture including lack of I-bands, decreased mitochondrial numbers and changes in mitochondrial organization, size and shape. Interestingly, these phenotypes were only observed in female animals. We also assessed the activity of specific respiratory chain complexes in situ using skeletal muscle fibers isolated from WT and KLF10 KO mice. These studies revealed a significant decrease in respiration rates for complexes I, II and IV in KLF10 KO mice compared to WT littermates. Histochemical analysis of critical muscle related enzymes revealed a nearly complete lack of succinate dehydrogenase activity in KLF10 KO muscle. This enzyme is responsible for conversion of succinate to fumarate and its decreased activity in KLF10 KO muscle correlates well with the accumulation of succinate observed following knockdown of KLF10 in C2C12 cells. Phenotypically, treadmill studies revealed that female KLF10 KO mice, but not male mice, exhibit an early onset of exercise fatigue compared to WT controls. Taken together, these data implicate novel roles for KLF10 in regulating mitochondrial complex activity and function as well as skeletal muscle differentiation and metabolism.