The in vivo anti-fatigue activity of the total flavonoids from sweet potato [Ipomoea batatas (L.) Lam.] leaf was investigated in male Kunming mice. The total flavonoids from sweet potato leaf (TFSL) were orally administered at doses of 50, 100 and 200 mg/kg for 4 weeks and the anti-fatigue effect was studied using a weight-loaded swimming test, along with the determination of serum urea nitrogen (SUN), blood lactic acid (BLA) and hepatic and muscle glycogen contents. The results showed that TFSL had significant anti-fatigue effects. TFSL extended the exhaustive swimming time, effectively inhibited the increase of BLA, decreased the level of SUN and increased the hepatic and muscle glycogen content of mice. Thus, TFSL may have potential as an anti-fatigue agent.