Gut microbiota influence the development of several chronic diseases including obesity, diabetes, and allergies. Recent studies suggest that an imbalance of gut microbiota may also influence muscle metabolism and contribute to muscle atrophy. This study focused on characterizing the impact of gut microbiota depletion on skeletal muscle by analyzing mice treated with gut-microbiota depleting antibiotics (ABT), mice treated with antibiotics followed by natural reseeding of microbiota (NAT), and control mice (CTL). Each group underwent running tests, where no differences were found in maximal aerobic velocity between each group. For the “Limit time to exhaustion during submaximal running test”, Tlim was significantly lower at day 9 than day 0 in both ATB and NAT mice. However, Tlim at day 19 was lower than day 0 in only the ATB group. Ex vivo contractile tests were then conducted using Aurora’s 305C Dual-Mode Muscle Lever and 701C High-Power Stimulator. EDL maximal strength was unaffected by the running tests; however, EDL muscle fatigue index was significantly reduced in ATB mice when compared with CTL and NAT mice. To investigate the role of gut microbiota on muscular glucose homeostasis, the authors analyzed markers linked to glucose metabolism in the gut-skeletal muscle. Levels of fasting-induced adipocyte factor (Fiaf) were significantly higher in the ATB group. In addition to this, free fatty acid receptor 3 (Gpr41) expression decreased. Following natural reseeding of the NAT group, increased levels of Gpr41 mRNA were observed when compared with the ABT group. Ileum muscle glycogen was also found to be significantly lower in ATB mice. These results highlight the interplay between gut microbiota and skeletal muscle. This study also provides insight into microbiome-based strategies for muscle therapy.