Hiccup reflex is a coordinated motor activity that causes a brief strong inspiratory movement accompanied by glottic adduction. Our previous study has demonstrated that mechanical stimulation of the dorsal epipharynx elicits hiccup-like response. To identify the afferent pathway of the hiccup-like response, the pharyngeal branch of the glossopharyngeal nerve (PB-GPN) which distributed to the dorsal epipharyngeal area was electrically stimulated in anesthetized, spontaneously breathing cats. To access the epipharynx and to directly observe a glottic movement, we made a submental opening at the region rostral to the epiglottis. An activity from the lateral cricoarytenoid (LCA) muscle of the larynx was recorded as an index of glottic adduction, and intrapleural pressure (Ppl) as an index of an inspiratory movement. Electrical stimulation of PB-GPN evoked a fixed motor pattern of hiccup-like response representing a spiky strong negative change in Ppl accompanied by an initial brief burst of LCA electromyogram (EMG). LCA excitation occurred prior to the spiky inspiratory movement. An initial and transient glottic adduction during the response was confirmed by direct observation. Electrical stimulation of the main trunk of the glossopharyngeal nerve evoked expiratory reflex, but not inspiratory (hiccup-like) response. These results indicated that PB-GPN is responsible for hiccup reflex.