Withania somnifera (Ashwagandha, Indian ginseng) roots powder has been studied that it acts as an antidepressant on central nervous system. However, the mechanisms underlying its antidepressant effect is poorly understood. NO, neuromodulator produced by nNOS from its precursors and like L-Arginine can be inhibited by its inhibitors like L-Cystenine. To elucidate the mechanism of Withania somnifera, we investigated the effect of a powdered root of Withania somnifera (WS) on nitric oxide (NO) production in Wistar rats. We found that WS produced a significant and concentration-dependent increase in NO production. Biochemical data from Lowry's method gave the protein content and NOx assay gave the nitrate/nitrite content during restraint stress. The results indicate a possible involvement of NO in stress-induced neurobehavioral effects.