To clarify the antinociceptive mechanism of acupuncture on acute pain, c-fos protein (Fos) expression induced by tooth pulp stimulation was immunohistochemically examined in the spinal trigeminal subnucleus pars caudalis (spVc) and the periaqueductal gray (PAG) of rats with or without Neiting acupuncture. The central projection of trigeminal ganglion neurons innervating in the tooth pulp was examined by tract-tracing method with horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP). Central terminals from the first maxillary molar tooth were labeled transganglionically in the dorsomedial part of spVc with WGA-HRP. Numerous numbers of Fos-immunoreactive (Fos-ir) cells were found in the spVc and PAG by stimulation of the tooth pulp with acetic acid or saline. Neiting acupuncture significantly reduced the Fos expression in the spVc induced by tooth pulp stimulation. On the other hand, Neiting acupuncture evoked many Fos-ir cells in the PAG. The present results suggest that Neiting acupuncture activated PAG neurons that sent descending inhibitory fibers to medullo-spinal nociceptive neurons, and reduced the number of Fos-expressed neurons in the trigeminal subnucleus pars caudalis mediating noxious information from teeth to the higher central nervous system.