Background <p>The neuronal activities within the dorsal raphe nucleus (DRN) and the ventrolateral orbital cortex (VLO) are strongly implicated in the development of depression, a condition comorbid with chronic pain. The goal of the present study was to determine whether and how the DRN-VLO pathway mediates chronic pain-induced depression.</p> Methods <p>Trigeminal neuralgia was induced unilaterally by chronic constriction injury of the infraorbital nerve. Depressive-like behaviors were assessed by the open field test, forced swimming test and tail suspension test. Neuronal projection tracing, chemogenetic manipulations and pharmacological interventions were performed to determine the DRN-VLO pathway projection activity and pathway-mediated anti-depressant mechanism in mice with trigeminal neuralgia. The neuronal activity was assessed by measuring the c-Fos level using immunofluorescence imaging.</p> Results <p>The VLO receives direct projection of the serotonergic neurons from the DRN. Anterograde or retrograde activation of the DRN-VLO pathway consistently produced anti-depressant effects in mice with neuropathic pain, whereas sustained inhibition of this pathway in healthy mice induced depressive-like behaviors. Activation of the 5-HT1A or 5-HT2A receptor in the VLO produced anti-depressant effects. Activation of the GABA_A receptors in the VLO weakened 5-HT1A receptor-mediated anti-depressant effect.</p> Conclusions <p>The present study has revealed that activation of the DRN-VLO pathway exerts an anti-depressant effect in mice with neuropathic pain, through stimulating the 5-HT2A receptors in the excitatory neurons and also the 5-HT1A receptors in the GABAergic interneurons via a dis-inhibition mechanism, to enhance neuronal activity of the VLO.</p>