Velvet antler (VA) is crucial and precious nourishment in China and some countries in Southeast Asia and has excellent anti‐fatigue effect. The incidence of fatigue syndrome has increased gradually. VA can be a potential source of anti‐fatigue products. Therefore, we investigated the anti‐fatigue activity of different sections (upper, middle, and basal section) of VA from different species (red deer and sika deer) via loading swimming test in mice. Furthermore, nucleosides are one kind of active components in VA which could considerably reduce fatigue in mice. In order to explore whether the nucleosides are correlated with anti‐fatigue effect, the contents of eight nucleosides (uracil, cytidine, hypoxanthine, xanthine, thymine, inosine, guanosine, and adenosine) were determined simultaneously using high‐performance liquid chromatography. The results indicated that the swimming time in mice was increased from basal to upper section, which was consistent with the change trend of the total contents of eight nucleosides of VA. Therefore, we speculated that the contents of nucleosides in VA may affect its anti‐fatigue effect. Furthermore, the contents of nucleosides were also used as a reference for evaluating the quality of different parts of VA obtained from red and sika deer.