Abstract: The mice were randomly divided into normal control group (physiological saline), Rhodotorula mucilaginosa polysaccharide high dose group (100 mg/kg·bw), medium dose group (50 mg/kg·bw), low dose group (25 mg/kg·bw), continuous intragastric administration after 10 days, the swimming time of mice was recorded by exhausted swimming test, and the level of serum urea nitrogen (BUN), blood lactate (BLA)and lactate dehydrogenase (LDH)levels and the determination of malondialdehyde in the liver (MDA), superoxide dismutase (SOD), catalase (CAT)and glutathione peroxidase (GSH-Px). The aim was to investigate the antifatigue effect of extracellular polysaccharide from Rhodotorula mucilaginosa. The results showed that extracellular polysaccharide could significantly prolong the swimming time of mice (p<0.01). Compared with the normal group, serum BUN level decreased significantly (p<0.01 for high dose group, p<0.05 for medium and low dose groups), BLA content also decreased significantly (p<0.01 for high dose group, p<0.05 for medium dose group), LDH activity was significantly increased (p<0.05 for high dose group), the content of the MDA in liver decreased significantly (p<0.05 for high, medium and low dose groups), SOD activity increased significantly (p<0.01 for high dose group, p<0.05 for middle dose group), CAT activity was significantly increased (p<0.01 for high, medium and low dose groups)and GPH-Px activity was significantly increased (p<0.01 for high dose group, p<0.05 for medium and low dose groups). Rhodotorula mucilaginosa extracellular polysaccharide had anti-fatigue effect.