Abstract: This study aimed to investigate the anti-fatigue effects of Lactobacillus fermentum FUA033 in mice and elucidate the underlying mechanisms. Forty male C57BL/6N mice were randomly assigned to four groups: control, low-, medium-, and high-dose L. fermentum FUA033. The probiotic was administered daily for four weeks, followed by assessments of endurance capacity, muscle mass, anti-fatigue biomarkers, energy metabolism, antioxidant activity, and gut microbiota composition. The results revealed a dose-dependent anti-fatigue effect of L. fermentum FUA033. Compared to the control group, the high-dose group exhibited a 3.35-s prolongation in exhaustive swimming time, accompanied by a 31.70% reduction in blood lactate and a 40.19% decrease in blood urea nitrogen levels. Hepatic glycogen, muscle glycogen, and triglyceride levels increased by 20.83%, 49.52%, and 62.50%, respectively. The antioxidant capacity was significantly enhanced, with increased superoxide dismutase, catalase, and glutathione peroxidase activities increasing by 49.29%, 24.21%, and 25.40%, respectively. Histological analysis revealed marked improvements in exercise-induced structural muscle abnormalities, including reduced tissue discontinuity and fiber disorganization. Furthermore, L. fermentum FUA033 administration increased the relative abundance of beneficial gut microbiota, particularly Bacteroidota (14.59% increase). These findings suggest that L. fermentum FUA033 possesses potent anti-fatigue properties and is a promising therapeutic probiotic.