Neuropathic pain-like hypersensitivity evoked by peripheral nerve injury is a salient clinical feature of pathologic pain; however, the underlying mechanisms of this condition remain largely unknown. Previous work has confirmed that spinal macrophage migration inhibitory factor (MIF) contributes to the pathogenesis of formalin-induced inflammatory hyperalgesia, but the role for MIF in neuropathic pain is still not well defined. After approval by the Ethical Committee of Animal Use and Care, the sciatic chronic constriction nerve injury-induced rodent model of neuropathic pain was built. The mechanical threshold with von Frey hairs and thermal latency with hot plate were measured, and the expression of spinal MIF, CD74, and downstream extracellular signal-regulated kinase 1/2 signaling cascade was detected. Finally, MIF gene mutation and exogenous recombinant MIF were used for further clarification. Intrathecal MIF tautomerase inhibitor reversed sciatic chronic constriction nerve injury-induced pain behaviors. The expression of MIF and CD74 up-regulated in a time-dependent manner in the ipsilateral spinal cord dorsal horn. These changes were associated with the activation of extracellular signal-regulated kinase 1/2 signaling by MIF/CD74 interaction, which subsequently led to up-regulation of interleukin-8 and N-methyl-D-aspartic acid receptor expression and additional production of prostaglandin E(2). Further, MIF gene mutation and exogenous recombinant MIF could desensitize and mimic sciatic chronic constriction nerve injury-evoked pain responses and cellular changes, respectively. MIF-associated extracellular signal-regulated kinase 1/2-N-methyl-D-aspartic acid receptor or prostaglandin E(2) cascade accounts for the changes in peripheral nerve injury-induced nociceptive responses.