Prolonged high-intensity exercise consumes significant energy, leading to fatigue and decreased performance. This study explores the effects of Bifidobacterium adolescentis CCFM1066 on exercise performance, gut microbiota, and its metabolites in mice. The results of the mouse experiments showed the mice which were intervened by Bifidobacterium adolescentis CCFM1066 have a significant increase in exercise performance, including forceful swimming time, fatigue baton turning time, and forelimb grip strength. Through metagenomic sequencing and differential metabolites, analysis indicated that the intervention of CCFM1066 increased Lachnospiraceae bacterium, Parabacteroides goldsteinii, Bacteroides xylanisolvens, and Bifidobacterium adolescentis and altered the key metabolic pathways including protein digestion and absorption and biosynthesis of amino acids. Supplementation with CCFM1066 modulates the production of short-chain fatty acids (SCFAs) and fatty acid amides (FAAs) by gut microbiota, decreasing levels of lactic acid (LA), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and creatine kinase (CK) while increasing muscle and hepatic glycogen content, thus reducing central nervous system fatigue and thereby improving exercise endurance and performance. These findings provide new insights into nutritional interventions for sports performance.