Background: Prior studies have revealed that crude Vicatia thibertica polysaccharide have the anti-fatigue effect, but the anti-fatigue effect and potential mechanism of purified polysaccharides endure blurred. Objectives: We intended to purify and gauge the anti-fatigue effects of a polysaccharide isolated from Vicatia thibertica polysaccharide 1 (VTP1). Materials and Methods: First, the exercise fatigue model was recognized in Kunming mice. After 14 days of continuous intragastric administration of VTP1 (50, 100, and 200 mg/kg/day), we assessed the anti-fatigue and antioxidant effects of VTP1 on fatigued mice. In addition, the effects of VTP1 on mitochondrial morphology of skeletal muscle of fatigue mice were detected by transmission electron microscopy. Meanwhile, the expression of mitochondrial DNA was noticed. Finally, using Western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction, respectively, to sense the expression of sirttuin1, peroxisome proliferative activated receptor-γ coactivator 1α (PGC-1α), and nuclear respiratory factor 1 (NRF1). Results: VTP1 meaningly extends the time to exhaustion during weight-bearing swimming and improves the endurance and bodyweight of fatigued mice. Furthermore, VTP1 exerts antioxidant effects, reduces creatine kinase, blood urea nitrogen and lactate dehydrogenase in the serum, surges superoxide dismutase and cuts malondialdehyde in the liver. Meanwhile, VTP1 diminishes degeneration of mitochondria in the skeletal muscle of mice with exercise fatigue and improves the biosynthetic efficiency of mitochondria in skeletal muscle. Finally, VTP1 can upregulate the mRNA and protein expression of PGC-1α and NRF1. Conclusion: VTP1 has an anti-fatigue effect, and the effect may be mediated by the PGC-1α/NRF1 signaling pathway.