Silymarin has been used for centuries for its hepatoprotective properties. The specific objective of this study was to evaluate the anti-fatigue properties of silymarin. The silymarin was administered orally at doses of 50, 100, and 200 mg/kg for 4 weeks; the fatigue level and exercise performance were evaluated using exhaustive swimming time and pole-climbing time, as well as levels of plasma lactate, ammonia, glucose, creatine kinase (CK), serum urea nitrogen (SUN), blood lactic acid (BLA), muscle glycogen (MG), and liver glycogen (LG) contents after an intensive swimming session. The results demonstrated that silymarin treatment decreased the BLA and SUN levels while increased the LG and MG levels. In addition, silymarin decreased plasma lactate and ammonia levels and CK activity after swimming test, this is related to the mechanism that increases energy storage (as glycogen) and release (as blood glucose), and decreases plasma levels of lactate, ammonia, and CK. The observation of the skeletal muscle structures of mice also confirmed that skeletal muscles became more damaged in the control group compared with the silymarin-treated mice after prolonged endurance exercise. Therefore, it is reasonable to infer that silymarin may bear potential pharmacological effects in combating fatigue.