Comorbid chronic neuropathic pain and anxiety is a common disease that represents a major clinical challenge. The underlying mechanisms of chronic neuropathic pain and anxiety are not entirely understood, which limits the exploration of effective treatment methods. Glutamatergic neurons in the ventrolateral periaqueductal gray (vlPAG) have been implicated in regulating pain, but the potential roles of the vlPAG in neuropathic pain-induced anxiety have not been investigated. Herein, whole-cell recording and immunofluorescence showed that the excitability of CamkIIα neurons in the vlPAG (vlPAG neurons) was decreased in mice with spared nerve injury (SNI), while electroacupuncture (EA) activated these neurons. We also showed that chemogenetic inhibition of vlPAG neurons resulted in allodynia and anxiety-like behaviors in naive mice. Furthermore, chemogenetic activation of vlPAG neurons reduced anxiety-like behaviors and allodynia in mice with SNI, and EA had a similar effect in alleviating these symptoms. Nevertheless, EA combined with chemogenetic activation failed to further relieve allodynia and anxiety-like behaviors. Artificial inhibition of vlPAG neurons abolished the analgesic and anxiolytic effects of EA. Overall, our study reveals a novel mechanism of neuropathic pain-induced anxiety and shows that EA may relieve comorbid chronic neuropathic pain and anxiety by activating vlPAG neurons.