The purpose of this study was to characterize the putative anxiolytic-like activity of an ethanolic extract prepared from the roots of Vitex negundo (VN) using the elevated plus maze (EPM) and light–dark exploration test in mice. Male mice were either treated orally with the VN extract or the positive control diazepam, respectively, 1 hour before behavioral evaluation. Oral administration of 100 and 200 mg/kg of VN extract significantly (P > 0.01) increased the percentage time spent on and the number of entries into the open arms of the EPM. The effect was comparable to that of the benzodiazepine diazepam (2 mg/kg p.o.). In light–dark exploration test, diazepam-treated rats significantly increased the time spent in light arena and decreased the duration of immobility, while VN treated rats also showed a significant (P >0.01) increase in the time spent (100 and 200 mg/kg) in light arena. Diazepam and the VN extracts do not produced any overt motor dysfunction. These results indicate that VN is an effective anxiolytic agent. In conclusion, the action of extract upon the anxiety models tested are in accord with the traditional use of VN L. and could be useful in primary medical care. Key words: Vitex negundo, anxiolytic, elevated plus maze, light–dark exploration, locomotor activity