Corneal sensory nerves have crucial roles in tear secretion and corneal wound healing in addition to the induction of ocular surface sensation. However, the regulatory system of corneal nerves is not fully understood. This study aims to examine the cholinergic regulation of trigeminal neurons innervating the cornea. In guinea pigs, corneal trigeminal neurons were labeled by treating the corneal surface with the retrograde dye FM1-43. Four days later, primary cultures of trigeminal ganglion neurons were made from the animals under anesthesia. Calcium imaging with Fura-2 was performed 24-48 hours after the dissociation. Perfusion of nicotine (1-100 µM), but not pilocarpine, concentration-dependently elevated intracellular Ca²⁺ concentration in a part of trigeminal neurons; the ratio of nicotine-sensitive neurons was 61% in FM1-43 positive corneal neurons, whilst being 38% in FM1-43 negative non-corneal ones. Compared among the populations classified with responsiveness to agonists of TRPM8, a cold sensor, and TRPV1, a polymodal nociceptor, a population showing the highest ratio of nicotine-sensitive corneal neurons was characterized by TRPM8 agonist-responsive and TRPV1 agonist-unresponsive. These findings suggest that nicotinic receptors might regulate the excitabilities of corneal sensory neurons, especially cold-sensitive ones.