Mulberry (Morus alba L.) is a tree species of Moraceae widely distributed in Southern China. In the present study, the white crystal of γ-aminobutyric acid (GABA) was purified from mulberry leaves, and its bioactivity was also investigated. The main results were as follows: first, the crude GABA was extracted from mulberry leaves by using biochemical methods. Then, the crude was purified by chromatography over an S-8 macroporous resin, Sephadex G-10, and 732 cation exchange resin to yield a white crystal. Lavage administration and exposure of GABA to male NIH mice showed no adverse effects on their growth and development. In an endurance capacity test, the average loaded-swimming time of medium dose was 111.60% longer than the control (P < 0.01). Further investigations showed that relative to that of model control, the respective blood lactate (BL) concentrations of low- and medium-dose were 28.52% and 28.81% lower (P < 0.05), whereas the levels of blood urea nitrogen (BUN) were 36.83% and 40.54% lower (P < 0.05), and that of liver glycogen (LG) levels were 12.81% and 17.22% lower (P < 0.05). The results indicated that GABA has an advantage over taurine of anti-fatigue effect. These findings were indicative of the anti-fatigue activity of GABA.