Introduction:Ziziphora Clinopodioides has been used widely for various therapeutic purposes in Iranian folk medicine. The current study aimed to determine interaction of antinociceptive effect of the Essential Oil of Ziziphora Clinopodioides (EOZC) and opioidergic system in male rats using formalin test.Methods:Sixty-four male Wistar rats were divided into eight groups. The groups 1 to 7 were injected with normal saline, vehicle (Tween-80, 0.5%), 10, 20, 40 mg/kg of the EOZC, morphine (5 mg/kg) and naloxone (2 mg/kg), respectively. Thirty minutes later, the formalin test was performed by intraplantar injection of formalin (50 μL, 2%). In group 8, naloxone (2 mg/kg) was injected 15 min before injection of EOZC (20 mg/kg), followed by formalin at 15 min later. The formalin test was done as time spent for licking and biting of the injected paw. Formalin induced a biphasic pain reaction. The chemical composition of EOZC was identified using Gas Chromatography-Mass Spectrometry (GC-MS).Results:EOZC (10, 20, and 40 mg/kg) dose dependently and morphine (5 mg/kg) reduced pain responses in the both phases of pain (P<0.05). Naloxone (2 mg/kg) alone had no effect on the severity of pain (P>0.05) but pretreatment with naloxone inhibited EOZC-induced antinociception activity (P<0.05). Based on the GC-MS results, EOZC comprised 65.22% carvacrol, 19.51% thymol, 4.86% p-cymene and 4.63% γ-terpinene.Conclusion:These results demonstrate that EOZC has antinociceptive effect and this effect might mediate via opioidergic pathways.