Background: The content and anti-fatigue effect of the purified anthocyanin from purple Daucus carota(PAPD) were studied. Materials and Methods: The content of total anthocyanins in PAPD was determined by the pH trial-difference method, and the content of cyanidin-3-o-glucoside in PAPD was determined by high-performance liquid chromatography (HPLC) method. Hundred ICR mice were randomly divided into five groups with 20 mice in each group: Negative control (NC) group, positive control (PC, red bull drink of 1.0 g/kg BW) group, PAPD dose group of low, middle, high (Low, 0.2 g/kg; Mid, 0.4 g/kg; Hig, 0.8 g/kg). Mice were intragastric injected continuously for 4 weeks; once a day, the NC group received an equal volume of saline, the PC group was given 1.0 g/kg BW red bull drink. After the last administration, the time of weight-bearing exhaustion swimming of 10 mice in each group was determined, the contents of lactic acid and urea nitrogen in the serum and glycogen in the liver and muscle tissues after 30 min of no-weight-bearing swimming of ten mice in each group were determined and the weight of mice during the experiment was also determined and the pathological analysis of the liver and kidney tissues in mice was conducted. Results: The content of total anthocyanin in PAPD was determined by the pH-differential method to be 132.46 mg/g DW, which was converted into fresh weight as 1.146 mg/g. The content of cyanidin-3-O-glucoside in PAPD was determined by HPLC, which was 31.45 mg/g DW, accounting for 21.37% of the total anthocyanin. Compared with the NC group, the exhausted swimming time of the PC group and PAPD group was significantly prolonged (P < 0.05), the contents of muscle glycogen and liver glycogen in the tissues of mice were significantly increased (P < 0.05), the contents of lactic acid and urea nitrogen in the serum of mice were significantly reduced (P < 0.05), and the lesions of the liver and kidney tissues were enhanced to some extent. Conclusion: The content of anthocyanin is rich in PAPD and has an observable anti-fatigue effect; its potential mechanism could improve the metabolism of sugar and lipid in muscle and liver tissues, reduce the oxidative damage of cells, improve the adaptability and endurance of the body to strenuous exercise, and enhance the immunity and antioxidant ability of cells, to play an anti-fatigue role.