Background: P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. Methods: Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. Results: Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1β, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. Conclusions: We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. Significance: Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.