5-HT receptors (5-HTRs) are widely expressed in the central nervous system, including in the ventrolateral orbital cortex (VLO). The VLO is an important cortical component for pain processing. Brain 5-HTRs are implicated in both pro- and anti- nociceptive functions. However, the roles of 5-HTRs in the VLO in trigeminal neuralgia and neuronal synaptic function remain to be understood. We used chronic constriction injury of infraorbital nerve (IoN-CCI) model and shRNA mediated gene knockdown in mice to investigate the role of 5-HTRs in the VLO in trigeminal neuralgia. We found that knockdown of 5-HTRs in the VLO aggravated spontaneous pain and mechanical allodynia in mice after IoN-CCI. At the synaptic level, decreasing 5-HTR expression by shRNA or inhibition of 5-HTR activity by its antagonist ketanserin decreased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) of the neurons in the VLO, whereas 5-HTR partial agonist 2,5-Dimethoxy-4-iodoamphetamine (DOI) enhanced sEPSCs of the neurons in the VLO. In summary, 5-HTRs in the VLO modulate the trigeminal pain by regulating neuronal glutamatergic activity.