Cinobufagin (CBG) has been shown to have antinociceptive properties. Nevertheless, the antinociceptive effect and mechanism of CBG are still unclear. The present study was designed to investigate the antinociceptive effect of CBG in the thermal and chemical pain models and further to explore the molecular target and potential signal pathway. As shown in the hot-plate test, formalin test, and acetic acid writhing test in mice, administration of CBG produced significant antinociceptive activity in a dose-dependent manner, and the antinociceptive effect was blocked by intraperitoneal pretreatment of methyllycaconitine citrate (an α7 nicotinic receptor antagonist) and intrathecal delivery of an α7 nicotinic receptor antagonist siRNA (α7-siRNA). Immunofluorescence demonstrated that the α7 nicotinic receptor and IκB/NF-κB were coexpressed in primary cultured lumbar DRG neurons. In the chemical pain models and primary cultured DRG neurons, Western blot analysis showed that the formation of p-IκB and p-NF-κB was regulated by CBG, and the effect of CBG was inhibited by α7-siRNA, and ELISA analysis indicated that CBG also regulated the expression of inflammatory cytokines through the α7 nicotinic receptor in DRG. These results suggest that CBG may activate an α7 nicotinic receptor, thereby triggering the inhibition of the DRG NF-κB signaling pathway, resulting in an antinociceptive effect in mice.