Neuropathic pain such as that seen in diabetes mellitus, results in part from central sensitisation in the spinal cord dorsal horn. However, the mechanisms responsible for sensory neuron activation remain unclear. There is evidence that disturbances in the integrity of the vascular network can be a causative factor in the development of neuropathic pain through alterations in the microenvironment of the dorsal horn sensory neuron. Here we show that induction of hypoxia at the level of the dorsal horn leads to the onset of pain hypersensitivity.  All animal experimentation was performed in line with the ARRIVE guidelines, with experimental procedures reviewed by the local Animal Welfare and Ethics Review Boards and performed in accordance with the UK Home office animals (Scientific procedures) Act 1986 and EU Directive 2010/63/EU. Using adult (30g) C57.bl6 male mice, nociceptive behavioural assays (von Frey hair – mechanical hypersensitivity and Hargreaves test – heat hypersensitivity) were performed with baseline nociceptive withdrawal thresholds determined prior to drug administration. Mice were treated with either intrathecal injection (under recovery gaseous anaesthetic isoflurane ~2% O2) of vehicle control (PBS; n=12) or 1mM Dimethyloxalylglycine (DMOG; n=12) and in an additional group intraperitoneal acetazolamide (ACZ; n=12). Nociceptive behavioural analysis (data provided as mean±SEM) was performed post intrathecal injection for upto 24 hrs, which demonstrated pronounced mechanical (Veh = 1.53g±0.04 vs DMOG=1.22g±0.05; ***p<0.001 One Way ANOVA) and heat hypersensitivity (Veh = 10.6s±1.1 vs DMOG=6.3s±2.2; ***p<0.001 One Way ANOVA) following DMOG administration. Lumbar spinal cord tissue was collected at 24hrs post DMOG administration demonstrating induced increased hypoxia signalling in the dorsal horn, depicted by increased expression of hypoxia markers hypoxia inducible factor 1&#55349;&#57084; and carbonic anhydrase 7 as well as markers of neuronal excitation (increased fos expression) depicted by western blot (n=3 per group), immunohistochemistry (n=3 per group) and RNAseq (n=5 per group)analysis. This was corroborated by electrophysiological recordings of lumbar spinal cord tissue slices demonstrating increased dorsal horn sensory neuron activity following treatment to induce hypoxia signalling. Furthermore, inhibition of carbonic anhydrase activity through intraperitoneal injection of Acetazolamide in combination with DMOG treatment inhibited hypoxia induced mechanical (DMOG + ACZ = 1.59g±0.0.26; ***p<0.001 One Way ANOVA) and heat hypersensitivity (DMOG + ACZ =11.86s±2.84; ***p<0.001 One Way ANOVA). This investigation demonstrates that induction of a hypoxic microenvironment in the dorsal horn, is an integral process by which sensory neurons are activated to initiate neuropathic pain states.