Neuropathic pain is caused by somatosensory nervous system disorder which happens in patients with different diseases. Akt3 regulates innate immune function and plays a role in neuropathic pain pathogenesis in rats. MiR-20b-5p is a microRNA which has been suggested to inhibit Akt3 expression through directly targeting Akt3 mRNA. This research focused on miR-20b-5p function in neuropathic pain by Akt3 expression inhibition. Chronic constriction injury (CCI) was employed to induce neuropathic pain in rats. Paw withdrawal thresholds and paw withdrawal latency were examined to show neuropathic pain development. Expression levels of relative genes or microRNA were checked using qRT-PCR and western blot. Inflammation cytokine levels were measured by enzyme-linked immunosorbent assay kits. In CCI rat model, miR-20b-5p level was declined and Akt3 mRNA level was upregulated. MiR-20b-5p mimics suppressed the enhanced neuropathic pain, neuroinflammation, and Akt3 expression. MiR-20b-5p directly targeted Akt3 mRNA and downregulated the Akt3 expression in rat primary microglial cells. MiR-20b-5p inhibitory function in neuropathic pain was suppressed by the upregulation of Akt3 expression. This research illustrated that miR-20b-5p alleviated neuropathic pain through the inhibition of Akt3 expression in CCI rat model.