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% (P<0.001) reduction in blood lactate and a 40.19% (P<0.001) decrease in blood urea nitrogen levels. Hepatic glycogen, muscle glycogen, and triglyceride levels increased by 20.83% (P<0.001), 49.52% (P<0.001), and 62.50% (P<0.001), respectively. The antioxidant capacity was significantly enhanced, with increased superoxide dismutase, catalase, and glutathione peroxidase activities increasing by 49.29% (P<0.001), 24.21% (P<0.001), and 25.40% (P<0.001), 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% increased). In conclusion, L. fermentum FUA033 alleviated exercise-induced fatigue in mice in a dose-dependent manner.