Frequent and persistent stressful events caused depressive illness. Stress is an aversive stimulus which disturbs physiological homeostasis and reflects a variety of biological systems. The present study was designed to investigate the nitric oxide mechanism in the protective effect of imipramine and venlafaxine against acute immobilization stress-induced behavioral and biochemical alterations in mice. Mice were immobilized for 6h. Imipramine (10 and 20mg/kg) and venlafaxine (5 and 10mg/kg) were administered 30min before subjecting the animals to acute stress. Behavioral tests (mirror chamber, actophotometer, tail flick test) and biochemical analysis (malondialdehyde level, nitrite, glutathione and catalase enzyme) were performed subsequently. Acute immobilization stress caused anxiety like behavior, analgesia, impaired locomotor activity and oxidative stress as compared to naive. Pretreatment with imipramine (10 and 20mg/kg) and venlafaxine (5 and 10mg/kg) significantly reversed immobilized stress-induced behavioral and biochemical alterations. l-arginine (100mg/kg) pretreatment with imipramine (10mg/kg) and venlafaxine (5mg/kg) significantly attenuated the protective effect of imipramine and venlafaxine. However, l-NAME (10mg/kg) and/or methylene blue (10mg/kg) pretreatment with lower dose of imipramine and venlafaxine significantly potentiated their protective effects which were significant as compared to their effect per se respectively. Present study highlights the involvement of nitric oxide mechanism in the protective effect of imipramine and venlafaxine against acute immobilization-induced behavioral and biochemical alterations in mice.