The comorbidity of anxiety and depression frequently occurs in patients with neuropathic pain. The ventrolateral orbital cortex (VLO) plays a critical role in mediating neuropathic pain and anxiodepression in rodents. Previous studies suggested that 5-HT receptors in the VLO are involved in neuropathic pain. Strong evidence supports a close link between 5-HT receptors and affective disorders such as depression and anxiety disorders. However, it remains unclear whether the 5-HT receptors in the VLO are involved in neuropathic pain-induced anxiodepression. Using a rat neuropathic pain model of spared nerve injury (SNI), we demonstrated that rats exhibited significant anxiodepression-like behaviors and the expression of VLO 5-HT receptors obviously decreased four weeks after SNI surgery. Microinjection of the 5-HT receptor agonist EMD-386088 into the VLO or overexpression of VLO 5-HT receptors alleviated anxiodepression-like behaviors. These effects were blocked by pre-microinjection of a selective 5-HT receptor antagonist (SB-258585) or inhibitors of AC (SQ-22536), PKA (H89), and MEK1/2 (U0126) respectively. Meanwhile, the expression of p-ERK, p-CREB, and BDNF in the VLO decreased four weeks after SNI surgery. Furthermore, administration of EMD-386088 upregulated the expression of BDNF, p-ERK, and p-CREB in the VLO of SNI rats, which were reversed by pre-injection of SB-258585. These findings suggest that activating 5-HT receptors in the VLO has anti-anxiodepressive effects in rats with neuropathic pain via activating AC-cAMP-PKA-MERK-CREB-BDNF signaling pathway. Accordingly, 5-HT receptor in the VLO could be a potential target for the treatment of the comorbidity of neuropathic pain and anxiodepression.