Regular exercise and balanced diet are the effective strategies to maintain healthy life style. Moderate exercise improves muscle metabolism, cardiovascular function and mitochondrial biogenesis by fatty acid utilization, enhancing mitochondrial metabolic rate and proliferation. Intense exercise can cause increased free radical production in the skeletal muscle and myocardium and forms muscle fatigue by increasing lactic acid content leading to muscle acidosis. Herbal extract supplementation has been suggested as an alternate therapy to treat fatigue induced stress. In the present study, we evaluated the anti-fatigue activity of Celastrus paniculatus seed extract (CPSE) in swimming endurance model of male BALB/C mice (25±2g). The work is an attempt of obtaining some clues to help elucidate the energy sensing, mitochondrial metabolism, angiogenesis, lactate and glucose transport mechanisms under swimming alone as well as in combination with 50mg/kg body weight CPSE administration by gavage. Here, we observed that CPSE supplementation enhances the swimming endurance by 2.5 folds. In order to understand the molecular mechanisms of exercise endurance activity we measured the exercise responsive skeletal metabolic regulators such as silent information regulator-1 (SIRT-1), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), AMP-activated protein kinase (AMPK) and lactate transporters monocarboxylic acid transporters-1 (MCT-1) and 4 (MCT-4), glucose transporter-4 (Glut-4), angiogenesis enhancer vascular endothelial growth factor (VEGF) and glycolysis regulator pyruvate dehydrogenase kinase-4 (PDK-4) and oxidative stress biomarkers superoxide dismutase-1 (SOD-1), catalase (CAT) and heat shock protein-70 (HSP-70). CPSE supplementation potentiated the expression of metabolic regulators. Further we used t-butyl hydroperoxide (t-BHP) induced C2C12 mice muscle cell model which mimics the reactive oxygen species (ROS) mediated fatigue stress to illustrate the underlying antioxidant and anti-apoptotic defense mechanism mediated by CPSE. Our observed results demonstrates that pre-treatment of C2C12 muscle cells with CPSE (50µg/ml) for 2h before 24h treatment with t-BHP protects cell damage by regulation of ROS formation by restoring the antioxidant enzyme/proteins SOD, CAT and HSP-70 levels. The extract also prevents t-BHP induced apoptosis by restoring the mitochondrial membrane potential (MMP) and inhibition of DNA damage. These results demonstrate that CPSE supplementation might be a valuable therapeutic approach in regulation of endurance activity as well as prevention of oxidative and apoptotic damage of cells.