The present study investigated anti-stress potential ofOcimum sanctumin chronic variable stress (CVS) paradigm. Further, the possible mechanism of anti-stress was exploredin vitrousing cell and cell-free assays. Rats were administeredO. sanctumfollowed by CVS regimen for a period of 16 days. On days 4, 8, 12, and 16, body weight and immobility time in forced swim test were measured. In addition, the possible inhibitory effect ofO. sanctumand ursolic acid on cortisol release and CRHR1 receptor activity were studied in cell-based assays, while inhibitory effects on 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and catechol-O-methyltransferase (COMT) were studied in cell-free assays. CVS group demonstrated less body weight gain and higher immobility time thanO. sanctumadministered groups, while oral administration ofO. sanctumsignificantly increased body weight gain and decreased the immobility time. Further,O. sanctumand its constituents inhibited cortisol release and exhibited a significant CRHR1 receptor antagonist activity. Also, they had specific inhibitory activity towards 11β-HSD1 and COMT activity. Thus,O. sanctumwas found to be effective in the management of stress effects, and anti-stress activity could be due to inhibition of cortisol release, blocking CRHR1 receptor, and inhibiting 11β-HSD1 and COMT activities. Copyright © 2016 John Wiley & Sons, Ltd.