BACKGROUND: Pain is an unpleasant experience that serves as a survival mechanism and is mediated via the central and peripheral nervous systems. Manna of hedysarum, also called Persian Manna or Taranjebin, is known to have medical properties in Persian traditional medicine. OBJECTIVES: The main purpose of the current paper is to determine the possible antinociceptive effect of Manna of hedysarum as well as the neurotransmitter systems involved, in mice. METHODS: The first experiment was designed to reveal the effective dose of Manna of hedysarum. Adult male albino mice were injected with Manna of Hedysarum (100, 200, and 400 mg/kg) Ethanolic Extract (MHEE) or morphine (5 mg/kg). In experiment 2, MHEE (400 mg/kg), naloxone (2 mg/kg), and MHEE (400 mg/kg) plus naloxone (2 mg/kg) were injected. Experiments 3-5 were similar to experiment 2, except that naloxone was replaced with L-NG-Nitro Arginine Methyl Ester (10 mg/kg), cimetidine (12.5 mg/kg), and cyproheptadine (4 mg/kg). Then the formalin test was performed, and the paw licking time was measured. RESULTS: Based on the findings, MHEE reduced the pain response in a dose-dependent manner (P<0.05). The co-injections of MHEE with mentioned antagonists significantly decreased the antinociceptive effect of MHEE on the licking and biting time of the injected paw (P<0.05). CONCLUSIONS: These findings suggest that MHEE has an antinociceptive effect in mice and possibly acts on opioidergic, nitrergic, histaminergic and serotonergic systems