Withanias omnifera (WS) is an Ayurvedic medicine, used for centuries as tonic, anxiolytic, antidepressant, analgesic, and adaptogen. Adaptogens are drugs promote non-specific resistance of the body and recognized as useful antistress agents. Although, the adaptogenic effect of WS is well documented, its mechanism is still not well defined. Stress is known to increase nitric oxide (NO) level in brain tissues, moreover, WS reported to inhibit nNOS expression in vitro. Hence, the present investigation was designed to evaluate the involvement of NO in adaptogenic effect of WS. Mice were exposed to overnight audiogenic stress from day 1 to 10. Various drugs treatments were given before the exposure on each day. Effects of stress were assessed in terms of anxiety by social interaction and depression by forced swim test. Results showed that exposure to audiogenic stress significantly induced anxiety and depression on day 1 and 4, whereas, no difference after day 7 and 10, indicated adaptation to stress. Administration of WS (50, 100 mg/kg, p.o.) attenuated the effect of audiogenic stress and facilitated the adaptation to stress. L-arginine impaired the adaptation to stress. In addition, concomitant administration of WS and L-NAME or 7-NI produced synergistic effect in audiogenic stress mice. Moreover, WS attenuated the effects of L-arginine. Further, WS administration significantly decreased audiogenic stress-induced increased NOx levels in cortex, hypothalamus and hippocampus. Thus, WS has significant adaptogenic activity and this effect is probably medicated through nitrergic system.