Withaferin-A dose-dependently (10 tob40 mg/kg) displayed anxiolytic activity, as measured by an increase in open arm exploration time in the elevated plus-maze (EPM), following intraperitoneal (i.p.) administration in rats. Acute administration of withaferin-A at 40.0 mg/kg significantly (P<0.05) increased open arm exploration time by 176% as compared to vehicle control, which is similar to the benzodiazepine diazepam at 1.0 and 3.0 mg/kg (191 and 200%, respectively). However, 24 h following sub chronic 5-day administration of diazepam twice daily (bid) at 3.0 mg/kg, diazepam was devoid of anxiolytic activity at 1.0 mg/kg, as measured by no difference in open arm exploration time when compared with vehicle control, while the 3.0 mg/kg dose still produced a significant (P<0.05) 175% increase in open arm exploration time. In contrast, following sub chronic administration of withaferin-A (40.0 mg/kg), a significant (P<0.01) enhancement in open arm exploration time was observed at 40.0 mg/kg (665% as compared to control). Therefore, withaferin-A resulted in anxiolysis which is similar to diazepam following acute administration in the EPM. However, following sub chronic administration unlike diazepam which showed an attenuation of anxiolytic activity, withaferin-A displayed enhanced anxiolytic efficacy and was devoid of tolerance. © 2011 Academic Journals.