Introduction: Pregabalin (PGB) is an analog of gamma-aminobutyric acid (GABA) with antinociceptive, antihyperalgesic and antiallodynic properties which frequently used in clinical pain management. Effect of PBG in neuropathic pain, incisional-inflammatory injury, post-operational pain, chronic pain and experimental pain models have already shown. It has been already known that muscarinic and serotonergic-2A receptors have a role in pain transmission. Methods: in this study, role of muscarinic and serotonergic-2A receptors in antinociceptive effect of pregabalin were evaluated with hot-plate and tail flick tests and effects of administered drugs on locomotor activity were measured with automated activity cage. Results: PGB treatment (30 and 100mg/kg) caused longer latency in hot plate and tail flick tests than saline group. That antinociceptive effect of pregabalin abolished by ketanserin (1mg/kg) and atropine (1mg/kg) treatment. Conclusion: However, there is lack of knowledge about role of nociceptive pathways underlying pregabalin mediated antinociception. Our results suggest that cholinergic and serotonergic systems have a role in antinociceptive effect of PGB which has seen in these somatic pain tests.