Background: Turnip (Brassica rapa L.) is a biennial plant, which belongs to Cruciferae Brassica, and is widely distributed at an altitude of 3000-4000 m in the Qinghai-Tibetan Plateau in western China. Turnip has been shown to have anti-hypoxia activities; however, there is limited scientific evidence of its anti-fatigue effects. Objectives: The objective is to evaluate the anti-fatigue properties and characterize the phytochemical compositions of turnip. Materials and Methods: Different proportions of turnip extract, including the total aqueous extract (50 mg/kg, per oral, [p. o.]), supernatant (50 mg/kg, p. o.), and polysaccharide (50 mg/kg, p. o.), were administered to mice, once a day for 20 days; following which, the exhaustive swimming time, liver and muscle glycogen, lactate dehydrogenase (LDH), creatine kinase (CK), blood lactic acid (BLA), and blood urea nitrogen (BUN) levels were tested. Ultra-high-performance liquid chromatography and Q-Exactive Orbitrap high-resolution mass spectrometry were used to analyze the turnip compounds. Results: Turnip, especially the supernatant portion, increased the exhaustive swimming time (476.01 ± 59.63 vs. 217.89 ± 32.70 s, P < 0.01), levels of liver glycogen (87.22 ± 16.28 vs. 18.53 ± 4.82 mg/kg, P < 0.01), and muscle glycogen (5.93 ± 0.51 vs. 2.56 ± 0.62 mg/kg, P < 0.01); decreased the activities of LDH (6,351.84 ± 344.81 vs. 7,359.54 ± 382.48 U/L, P < 0.01) and CK (974.66 ± 112.89 vs. 1,115.69 ± 113.52 U/L, P < 0.05); and decreased the levels of BLA (8.68 ± 1.86 vs. 10.99 ± 1.52 mmol/L, P < 0.05) and BUN (4.40 ± 0.44 vs. 5.21 ± 0.52 mmol/L, P < 0.01) when compared to the control group, and exhibited the best performance in relieving fatigue. Fourteen low-molecular-weight chemicals were detected in the supernatant portion, some of which had anti-fatigue effects. Conclusion: Our findings suggest that turnip prevents fatigue in mice and has the potential to be developed into health products to reduce fatigue.