The involvement of nitric oxide (NO) in stress-induced neurobehavioral changes in rats was evaluated using the elevated plus maze and open field tests. Restraint stress (1 h) reduced both the number of entries and time spent in open arms, with both expressed vehicle-controls and these changes were reversed with diazepam (1 mg/kg) and withaferin A (10 - 50 mg/Kg, i.p), N-nitro-L-arginine methyl ester (L-NAME) (50 mg/kg)pretreatment. In the open field test, the restraint stress-induced (a) increased entry latency and (b) decreased ambulation and rearing were reversed by diazepam and withaferin A(40 mg/Kg), N-nitro-L-arginine methyl ester (L-NAME) (50 mg/kg). Biochemical data showed that withaferin A had a direct effect on the total brain nitrate/nitrite content after acute restraint stress. The root extract of Withania somnifera (100 - 500 mg/kg, oral) also showed anxiolytic effects in dose dependent manner. We suggest that Withaferin A in doses of 40 and 50 mg/kg causes a mild anxiolytic-like behavior and discuss the possibility that this reported effect could be caused by inhibition of nitric oxide synthase. © 2010 Academic Journals.