Objective: To investigate the analgesic and anti-inflammatory property and possible involvement of opioid receptors of ethyl acetate extract from aerial parts of Daphne mucronata (D. mucronata) in mice by formalin test. Methods: Single doses of 2.5, 5.0 and 10.0 mg/kg of body weight of ethyl acetate extract of D. mucronata were intraperitoneally administered to the mice 30 min before analgesic test. The anti-nociceptive effect of preparations was evaluated based on the formalin in mice. Results: The results indicated that the extract (2.5, 5.0 and 10.0 mg/kg) increased the pain threshold of mice and induced analgesia in both phases of formalin test. Like morphine sulfate (5.0 mg/kg, i.p.), the extract also showed more effective analgesic effect on the late phase of formalin test. Pre-treatment of animals with naloxone (5.0 mg/kg i.p.) did not inhibit the effects of the extract. Conclusions: Our findings suggest that D. mucronata contains potential analgesic and anti-inflammatory compounds which support its traditional use. Moreover, it seems that the analgesic and anti-inflammatory effects of the extract is mediated by non-opioid mechanisms. Further pharmacological studies are required to determine whether the analgesic mechanisms are actually responsible for such properties.