Pharmacological effects of hydroalcoholic extract prepared from Hypericum origanifolium Willd. (Guttiferae) on behavioral parameters and pain perceptions of mice were investigated in this study. Depression, anxiety, spontaneous locomotor activity, and motor coordination parameters of mice were assessed by modified forced swimming, hole board, activity cage, and rota-rod tests, respectively. In addition, antinociceptive effect was evaluated by performing hot-plate, tail-clip, and formalin tests. Reboxetine (20 mg/kg), diazepam (1 mg/kg), and morphine (10 mg/kg) were used as reference antidepressant, anxiolytic, and analgesic drugs, respectively. Phytochemical analyses exhibited that chlorogenic acid (2317.12 ppm) and rutin (2108.79 ppm) were the main phenolic compounds in the H. origanifolium extract. The extract (50, 100, and 250 mg/kg) induced significant antidepressant, anxiolytic, and antinociceptive activities following the acute administrations. Anxiolytic effect was antagonized by flumazenil (a benzodiazepine receptor antagonist, 2.5 mg/kg, i.p.) pre-treatment, which indicated the participation of GABA(A)-benzodiazepine receptor complex in the activity. Moreover, centrally and peripherally mediated antinociception reversed by naloxone (a non-selective opioid receptor antagonist, 5 mg/kg, i.p.) pre-treatment, indicating the involvement of opioid system in the pharmacological action. These findings are the first to indicate the pharmacological effects of the H. origanifolium extract on the emotional state and pain perceptions of mice.