Withania somnifera (WS) possess anti-inflammatory and antioxidant properties. WS preparations have a potential therapeutic role in the central nervous system (CNS) related disorders in animal models. In this study, the possible protective effect of acute aqueous WS root extract on behavioral despair was explored and compared with fluoxetine, an antidepressant with selective serotonin (5-HT) reuptake inhibitor activity (SSRI). Further, the probable involvement of nitric oxide (NO) determined to measure immobility time in forced swimming test (FST) and tail suspension test (TST) in male mice. Immediately after assessment of locomotor activity, the immobility time was evaluated. WS was administered intraperitoneally (200, 400 mg/kg; i.p.) 60 min before the behavioral tests. To assess the involvement of NO in the possible protective effect of WS, a non-specific NO synthase inhibitor, NG-L-arginine methyl ester (L-NAME, 10 mg/kg, i.p.) was administered 30 min before the extract administration (400 mg/kg, i.p.), 90 min before the tests. Acute WS extract (200, 400 mg/kg, i.p.) dose-dependently decreased the immobility time in FST, P<0.05, P<0.001, respectively and 400 mg/kg proved the most effective dose and this dose was comparable to fluoxetine (20 mg/kg, i.p. WS (400 mg/kg, i.p.) also lowered the immobility measure in TST (P<0.05). However, these effects were not related to change in locomotor activity. Moreover, L-NAME (10 mg/kg, i.p.) did not influence the effect of the extract on the behavioral tests. As a consequence, the immobility time was virtually constant between the group received the extract (400 mg/kg) alone, and the group received L-NAME (10 mg/kg) before the extract. It is probable that NO does not mediate this beneficial effect, and WS may affect other neurochemical systems and pathways.