Tolerance to the analgesic effect of opioids is the major concern of long-term administration of these compounds. In this study, the effects of hydroalcoholic extract of Vitex agnus-castus (VAC) on the acquisition and expression of morphine-induced tolerance were evaluated in mice by tail-flick test. To evaluate the VAC effects on the development or expression of morphine tolerance, animals received VAC (i.p.), 30 min before morphine (50 mg/kg; s.c.) during induction period once daily for 3 days; or 30 min before challenge dose of morphine (5 mg/kg) before (day 1) and after morphine-induced tolerance (day 4), respectively. The analgesic effect of VAC was evaluated every 30 min till 2 h. VAC at the doses of 120 and 180 mg/kg could suppress the development of tolerance. In addition, VAC at the dose of 180 mg/kg attenuated the expression of morphine-induced tolerance. While, the VAC alone had no analgesic effect, its combination with morphine at the dose of 180 mg/kg, could significantly enhance the antinociceptive effect of morphine. VAC can attenuate the expression of morphine's tolerance. Based on the enhancement of the analgesic effect of morphine's challenge dose by VAC, it seems that the expression is more attenuated by additive effect of VAC than reversal of tolerance. The ability of VAC to ameliorate the acquisition and expression of morphine-induced tolerance in mice may be worthwhile for traditional chronic pains treatment in combination with opioids without concern of tolerance and dependence. © 2016, SILAE (Italo-Latin American Society of Ethnomedicine). All rights reserved.