Abstract: This study investigated the effects and potential mechanisms of Lactobacillus acidophilus XY27 (LAXY27) on enhancing exercise endurance in mice. The experiment used D-galactose induction to establish a mouse oxidative stress injury model, and systematically evaluated the effects of LAXY27 on the model animals. The results showed that after gavage of LAXY27H (1.5×10⁹ CFU/kg body weight), the exercise endurance of oxidative stress mice was significantly improved, manifested by an extension of swimming duration from 188 s to 301 s, and the improvement effect was significantly (P<0.05) better than that in the vitamin C control group (227 s) (150 mg/kg body weight oral administration). The concentrations of lactic acid (LA), blood urea nitrogen (BUN), and creatine kinase (CK) in the LAXY27H intervention group were significantly reduced (P<0.05) compared to the model group, while the levels of muscle glycogen (MG) and liver glycogen (HG) were significantly higher (P<0.05), indicating that it alleviated exercise-induced fatigue by optimizing energy metabolism. Pathological results showed that LAXY27 could alleviate liver oxidative stress damage. Molecular mechanism analysis showed that LAXY27 could up-regulate the mRNA expression of 5'-AMP-activated protein kinase α1 subunit (AMPKα1), AMPKα2, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), superoxide dismutase 2 (SOD2, Mn-SOD) and glutathione peroxidase 1 (GPx1) and down-regulate the expression of acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS) in the liver tissue of mice with oxidative stress injury. Meanwhile, LAXY27 had the same effect on mRNA expression in mouse gastrocnemian muscle, which could up-regulate the expression of AMPKα1, AMPKα2, PGC-1α, SOD2 and GPx1 in mice with oxidative stress injury. Intestinal microbial analysis showed that LAXY27 intervention significantly increased the abundance of Firmicutes, Lactobacillus and Bifidobacterium (P<0.05). At the same time, it inhibited the excessive proliferation of Bacteroidetes, further supporting its mechanism of improving host function through microbiota regulation. This study shows that LAXY27 can improve oxidative stress and exercise ability in mice through multi-target regulation, including activation of energy metabolism pathways, enhancement of antioxidant enzyme expression and optimization of intestinal flora, and its comprehensive intervention effect is better than that of the traditional antioxidant vitamin C.