Background: Neuropathic pain, a type of chronic pain as a result of direct central or peripheral nerve damage, is associated with significant quality of life and functional impairment. Its underlying mechanisms remain unclear. We investigated whether ROR2, a member of the receptor tyrosine kinase-like orphan receptor (ROR) family, participates in modulation of neuropathic pain. Methods: Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and von Frey filament testing. Immunofluorescence staining was used to detect expression of ROR2 in neuronal nuclei. Fos expression was determined by immunocytochemistry. Phosphorylation status was detected by western blot and immunoprecipitation. Small interfering RNA was used to knock down ROR2 expression. Results: ROR2 was upregulated and activated in spinal neurones after chronic constriction injury (CCI) in mice [1.3 (0.1) to 2.1 (0.1)-fold of sham, P<0.01] from Day 1e21. CCI induced significant demethylation of the CpG island in the ROR2 gene promoter [0.37 (0.06) vs 0.12 (0.03)% CpG methylation, P<0.001]. Knockdown of ROR2 in the spinal cord prevented and reversed CCI-induced pain behaviours and spinal neuronal sensitisation [Fos expression: 130 (12) vs 81 (8) cells, P<0.05; 120 (11) vs 70 (7) cells, P<0.05]. In contrast, activation of spinal ROR2 by intrathecal injection of Wnt5a induced pain behaviours and spinal neuronal sensitisation [Fos expression: 11 (1) vs 100 (12) cells, P<0.001] in wild-type mice. Furthermore, ROR2-mediated pain modulation required phosphorylation of N-methyl-D-aspartate receptor 2B subunit (GluN2B) at Ser 1303 and Tyr1472 by pathways involving protein kinase C (PKC) and Src family kinases. Intrathecal injection of GluN2B, PKC, or Src family kinase-specific inhibitors significantly attenuated Wnt5a-induced pain behaviours. Conclusions: ROR2 in the spinal cord regulates neuropathic pain via phosphorylation of GluN2B, suggesting a potential target for prevention and relief of neuropathic pain.