Previous studies have shown that astrocyte activation in the anterior cingulate cortex (ACC), accompanied by upregulation of the astrocyte marker S100 calcium binding protein B (S100B), contributes to comorbid anxiety in chronic inflammatory pain (CIP), but the exact downstream mechanism is still being explored. The receptor for advanced glycation end-products (RAGE) plays an important role in chronic pain and psychosis by recognizing ligands, including S100B. Therefore, we speculate that RAGE may be involved in astrocyte regulation of the comorbidity between CIP and anxiety by recognizing S100B. Here, we investigated the potential role of RAGE and the correlation between RAGE and astrocyte regulation in the ACC using a mouse model of complete Freund's adjuvant (CFA)-induced inflammatory pain. We detected substantial upregulation of RAGE expression in ACC neurons when anxiety-like behaviors occurred in CFA-treated mice. The inhibition of RAGE expression decreased the hyperexcitability of ACC neurons and alleviated both hyperalgesia and anxiety in CFA-treated mice. Furthermore, we found that the ACC astrocytic S100B level increased over a similar time course. Intra-ACC application of S100B or downregulation of ACC astrocytic S100B via suppression of astrocyte activation significantly affected RAGE levels and the relative behaviors of CFA-treated mice. Taken together, these findings suggest that the upregulation of ACC neuronal RAGE results from the activation of astrocytic S100B and leads to the maintenance of pain perception and anxiety in the late phase after CFA injection, which may partly explain the mechanism by which ACC neuron‒astrocyte coupling promotes the maintenance of CIP and anxiety comorbidity.