One distinguishing feature of primary afferent neurons is their ability to bind the lectin IB₄. Previous work suggested that neurons in the inner part of lamina II (IIi), onto which IB₄-positive sensory neurons project, facilitate nociceptive transmission following tissue or nerve injury. Using an IB₄-saporin conjugate (IB₄-SAP), we examined the contribution of IB₄-positive neurons to nociceptive processing in rats with and without nerve injury. Intrasciatic injection of IB₄-SAP (5 μg/5 μl) significantly decreased IB₄-labeling and immunoreactive P₂X₃ in the spinal cord and delayed the behavioral and neuroanatomical consequences of L5 spinal nerve ligation (SNL) injury. In the absence of injury, thermal and mechanical nociceptive thresholds increased 2 weeks post-treatment only in IB₄-SAP-treated, but not control (saline or saporin only), rats. Acute NGF-induced hyperalgesia was also attenuated following IB₄-SAP treatment. In the SNL model, mechanical allodynia failed to develop 1 and 2 weeks post-injury, but was fully established by 4 weeks. Moreover, neuropeptide Y immunoreactivity (NPY-ir), which increases in the spinal cord after nerve injury, was unchanged in IB₄-SAP-treated animals whereas immunoreactive PKCγ decreased 2, but not 4, weeks post-injury. Quantitative RT-PCR revealed a reduction in P₂X₃ mRNA in L4 DRG of IB₄-SAP-treated animals, but no change in TrkA expression. Our results suggest that IB₄-positive neurons in L4 are required for the full expression of NGF-induced hyperalgesia and participate in the behavioral and anatomical consequences that follow injury to the L5 spinal nerve.