Cannabidiol (CBD), a nonpsychoactive compound from Cannabis, has various bioactive functions in humans and animals. Evidence suggests that CBD promotes muscle injury recovery in athletes, but whether and how CBD improves endurance performance remains unclear. Here we investigated the effects of CBD treatment on exercise performance in mice and assessed whether this effect involves the gut microbiome. CBD administration significantly increased treadmill running performance in mice, accompanied by an increase in oxidative myofiber composition. CBD also increased mitochondrial biogenesis and the expression of associated genes such as PGC-1α, phosphorylated CREB and AMPK in muscle tissue. Interestingly, CBD altered the composition of the gut microbiome, and antibiotic treatment reduced the muscle endurance-enhancing effects of CBD and mitochondrial biogenesis. We isolated Bifidobacterium animalis, a microbe increased by CBD administration, and named it KBP-1. Treatment with B. animalis KBP-1 in mice resulted in improved running performance. Whole-genome analysis revealed that B. animalis KBP-1 presented high expression of genes involved in branched-chain amino acid biosynthesis, expression of branched-chain amino acid release pumps and metabolism of lactic acid. In summary, our study identified CBD and B. animalis KBP-1 as potential endurance exercise-promoting agents.