Mechanical allodynia, characterized by a painful sensation induced by innocuous stimuli, is thought to be caused by disruption in pain-related regions. Identification and reversal of this pathologic neuroadaptation are therefore beneficial for clinical treatment. Previous evidence suggests that 5-HT₆ receptors in the ventrolateral orbital cortex (VLO) are involved in neuropathic pain, but their function is poorly understood. The aim of the present study is to unveil the role of 5-HT₆ receptors in the VLO and the underlying mechanisms in pain modulation. Here, by using the spared nerve injury (SNI) pain model, first, we report that 5-HT₆ receptor protein decreased in the contralateral VLO compared with the ipsilateral VLO in rats with allodynia. Second, microinjection of the selective 5-HT₆ receptor agonists EMD-386088 and WAY-208466 into the contralateral VLO consistently and significantly depressed allodynia. Third, microinjection of the selective antagonist SB-258585 blocked the agonist-induced anti-allodynic effect, while the antagonist applied alone to the VLO had no effect. Furthermore, the anti-nociceptive effect of EMD-386088 on neuropathic pain was prevented by the adenylate cyclase (AC) inhibitor SQ-22536, and protein kinase A (PKA) inhibitor H89, suggesting that AC/PKA signaling might underlie the antinociception of agonists. Finally, the 5-HT₆ receptors were found to be colocalized with a glutamate transporter (EAAC1) by immunofluorescent staining, and the glutamate receptor antagonist kynurenic acid was found to completely block antinociception. These findings indicated that the antinociceptive effect of 5-HT₆ receptor agonists might occur via interaction with the glutamatergic system. Altogether, the agonists activated 5-HT₆ receptors present in the glutamatergic neurons in the VLO to facilitate the AC/PKA cascade, which subsequently might evoke glutamate release, thus depressing allodynia. These findings suggest a potential therapeutic role of 5-HT₆ receptor agonists in treating neuropathic pain.