Background: Glaucium vitellinum is an endemic species and is extensively exploited as an anti-inflammatory agent in Iranian traditional medicine. Objectives: This study was designed to evaluate the antinociceptive activities of G. vitellinum methanol extract in male mice. Materials and Methods:The formalin and hot-plate methods were used for pain evaluation in mice. Glaucium vitellinum extract (50, 100, 200 and 400 mg/kg body weight IP), saline and morphine (2 mg/kg, IP) were administered 15 minutes prior to the formalin test. The nociceptive responses were divided to two phases; phase І (0 -15 minutes) and phase ІІ (15 -60 minutes) were compared to the control and morphine. In the hot-plate test, G. vitellinum extract (80, 160, 200 and 250 mg/kg IP), saline and morphine (5 mg/kg, IP) were administered and, behavioral responses were immediately tested, 15, 30, 45 and 60 minutes after the injection. Comparisons between the groups were carried out using the analysis of variance (ANOVA), and post hoc Tukey's test. Results: All doses of G. vitellinum extract induced anti-nociception activity during the first and second phases of the formalin test. The extract showed a significant (P < 0.05) dose-related inhibition during the first phase compared to the control group. In the second phase of the formalin test, the extract showed analgesic activity comparable to the effect of morphine. In pre-treatment with non-selective opioid receptor antagonist, naloxone could reverse the anti-nociceptive effect of the extract in the formalin test. In the hot-plate method, with the highest dose of 250 mg/kg, the anti-nociceptive activity of the studied extract was comparable to the standard drug, morphine. Conclusions: This study revealed that G. vitellinum extract possessed a significant anti-nociceptive activity in formalin pain models and hot-plate test in mice and might have a potent role against pain.