Objective To investigate the central mechanism of glial cells of the dorsal reticular nucleus (DRt) in the modulation of chronic orofacial pain after removal of experimental occlusal interference(EOI) in rats. Methods Twenty-four male SD rats (180-200 g) were randomly divided into three groups (8 in each group): sham group, EOI hyperalgesia maintaining group (occlusal interference appliance removed on day 8 after wearing 0.4 mm thick crowns), EOI hyperalgesia maintaining+DRt damage group (EOI hyperalgesia maintaining rats were injected with ibotenic acid to damage DRt). The non-reflexive behaviors of the three groups were evaluated by using orofacial operant test on 7, 10, 14 d after model establishment. Nine male SD rats were randomly divided into three groups (3 in each group): sham group, EOI hyperalgesia maintaining group (day 8 after wearing 0.4 mm thick crowns, before removal of EOI), EOI hyperalgesia maintaining group 6 d (6 days after EOI removed on day 8). DRt sections were obtained and processed for immunofluorescence staining for glai fibrillary acidic protein (GFAP) and OX-42. The levels of expression were hemi-quantitatively analyzed to evaluate the fluorescence area and fluorescence intensity of astrocytes and microglia. Results EOI hyperalgesia maintaining group and EOI hyperalgesia maintaining+DRt damage group exhibited lower feeding time in orofacial operant test, which implied hyperalgesia (P<0.05). The hyperalgesia in EOI hyperalgesia maintaining group persisted after the removal of EOI, and the difference was statistically significant at 10 d and 14 d compared with sham group (P<0.05), while the hyperalgesia in the EOI hyperalgesia maintaining+DRt damage group showed a rebound trend, and the difference was not statistically significant at 10 d and 14 d compared with sham group (P>0.05). The total feeding time at 14 d significant longer compared with the EOI hyperalgesia maintaining group (P<0.05). Semi-quantitative analysis of immunofluorescence staining showed that the fluorescence area and fluorescence intensity of GFAP and OX-42 in EOI hyperalgesia maintaining group did not show any increase compared with that of sham group (P>0.05), whereas the fluorescence area and fluorescence intensity of GFAP as well as the fluorescence area of OX-42 in EOI hyperalgesia maintaining group 6 d were significantly higher (P<0.05). The fluorescence area and fluorescence intensity of GFAP and OX-42 in EOI hyperalgesia maintaining group did not show any increase compared with that of EOI hyperalgesia maintaining group 6 d (P>0.05). Conclusion DRt was involved in the persistent maintenance of hyperalgesia in the EOI model after removal of occlusal interference, in which astrocyte and microglia activation in DRt were the central mechanisms for the maintenance of hyperalgesia.