The main mechanism of action of herbicide Trifluralin is the inhibition of cell mitosis. As an important non-protein amino acid, γ-aminobutyric acid (GABA) contributes in certain physiological functions and stress tolerance. So, to detect the effect of Trifluralin and role of GABA on enzymatic activities and content of proline and growth substances including gibberellin (GA3) and cytokinin (6-benzyl amino purine) an experiment was conducted using squash (Cucurbita pepo) seedlings. Four levels (0, 5, 15 and 25 ppm) of trifluralin and presence or absence of GABA were applied in completely randomized design and in 3 replicates. Pots were sprayed by trifluralin 2 days after planting. Distilled water and GABA (500 μ mol l-1) were sprayed on control and treated plants respectively 3 days after planting. Effect of trifluralin on ornithine amino-transferase (OAT), proline dehydrogenase (PDH) and content of proline and also GA3 and 6-benzyl amino purine was significant (p≤ 0. 01) according to ANOVA. On the other hand, GABA had a significant effect on activity of PDH, content of proline and GA3 and cytokinin level. Content of proline and activity of OAT and the amounts of GA3 and cytokinin increased significantly, although PDH was decreased under influence of GABA. Content of proline and OAT activity were raised and GA3, benzyl amino purine and activity of PDH were declined by increased levels of trifluralin. Overall, it can be concluded that growth and biochemical indices of squash plants were improved by GABA under toxicity of herbicide trifluralin.