γ-Amino butyric acid (GABA) and proline play a crucial role in protecting plants during various environmental stresses. Their synthesis is from the common precursor glutamic acid, which is catalyzed by glutamate decarboxylase and Δ1-pyrroline-5-carboxylate synthetase respectively. However, the dominant pathway under water stress has not yet been established. To explore this, excised tobacco leaves were used to simulate a water-stress condition. The results showed GABA content was much higher than that of proline in leaves under water-deficit and non-water-deficit conditions. Specifically, the amount of GABA significantly increased compared to proline under continuous water loss for 16 h, indicating that GABA biosynthesis is the dominant pathway from glutamic acid metabolism under these conditions. Quantitative reverse transcription polymerase chain reaction and protein Western gel-blot analysis further confirmed this. To explore the function of GABA accumulation, a system producing superoxide anion (O2−), peroxide hydrogen (H2O2), and singlet oxygen (1O2) was employed to investigate the scavenging role on free-radical production. The results demonstrated that the scavenging ability of GABA for O2−, H2O2, and 1O2 was significantly higher than that of proline. This indicated that GABA acts as an effective osmolyte to reduce the production of reactive oxygen species under water stress.