In this study, the antifatigue effects of acupuncture had been investigated at the metabolic level on the young male athletes with exhaustive physical exercises. After a series of exhaustive physical exercises and a short-term rest, the athletes either were treated with needling acupuncture on selected acupoints (TA group) or enjoyed an extended rest (TR group). NMR-based metabolomics analysis was then applied to depict the metabolic profiles of urine samples, which were collected from the athletes at three time points including the time before exercises, the time before and after the treatment of acupuncture, or taking the extended rest. The results from multivariate statistical analysis indicated that the recoveries of disturbed metabolites in the athletes treated with acupuncture were significantly faster than in those only taking rest. After the treatment with acupuncture, the levels of distinguished metabolites, 2-hydroxybutyrate, 3-hydroxyisovalerate, lactate, pyruvate, citrate, dimethylglycine, choline, glycine, hippurate, and hypoxanthine were recovered at an accelerated speed in the TA group in comparison with the TR group. The above-mentioned results indicated that the acupuncture treatment ameliorated fatigue by backregulating the perturbed energy metabolism, choline metabolism, and attenuating the ROS-induced stress at an accelerated speed, which demonstrated that acupuncture could serve as an alternative fatigue-relieving approach.