Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5 μg/20 μl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10 min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60 min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200 ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100 ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100 ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200 ng/site atropine and mecamylamine (200 ng/site) prevented oxotremorine (100 ng/site)-, and epibatidine (100 ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity.