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BACKGROUD Moringa oleifera leaves are rich in nutrients and bioactive ingredients. This study was aimed to evaluate the anti-fatigue effect of the ethanol extract of Moringa oleifera leaves (MLEE) on mice and its primary mechanism of action using weight-loaded force swimming test. In the present study, MLEE was prepared by ultrasound-assisted extraction, and its anti-fatigue effect and antioxidant capacity were evaluated in mice. Mice were administrated MLEE (320 mg/kg·BW) for 15 days. RESULTS MLEE supplementation significantly increased levels of glucose and non-esterified fatty acids (NEFA), while decreased levels of lactate and blood urea nitrogen in serum (p < 0.05); the levels of glycogen in the liver and muscle were also increased, as was the activity of glycogen synthase and the level of NEFA in muscle (p < 0.05). According to a Western blot analysis, MLEE increased the expression of AMPK1, JNK, AKT, and STAT3 in the muscle of mice. CONCLUSIONS Our findings indicate that MLEE has an anti-fatigue effect via the AMPK-linked route, which enables it to control energy metabolism and enhance antioxidant enzyme activity. This article is protected by copyright. All rights reserved.