The present study aimed to determine the effects of polysaccharides from Gynostemma Pentaphyllum (PGP) supplementation on exhaustive exercise-induced fatigue in mice. The mice were divided into four experimental groups at random, i.e. control, low-dose PGP, medium-dose PGP, and high-dose PGP treatment groups. An oral administration of distilled water was given to the mice in the control group, and different doses of PGP (100, 200 and 400 mg/kg body weight/day) were given to the mice in the treatment groups. After 28 days, the weight-loaded swimming test was performed, along with the determination of blood biochemical parameters, liver glycogen and muscle glycogen contents. The results indicated that PGP supplementation had anti-fatigue effects, which could make mice swim for a longer time to exhaustion, with the blood lactic acid, serum urea nitrogen, serum triglycerides, and serum creatine kinase contents decreased, and the liver glycogen and muscle glycogen contents increased.