Extensive regeneration of sensory axons into the spinal cord can be achieved experimentally after dorsal root injury, but no effort has been made to target regenerating axons and restore a normal lamina-specific projection pattern. Ectopic axon growth is potentially associated with functional disorders such as chronic pain and autonomic dysreflexia. This study was designed to target regenerating axons to normal synaptic locations in the spinal cord by combining positive and negative guidance molecules. Previously, we observed that, after dorsal rhizotomy, overexpression of NGF leads to robust regeneration and sprouting of calcitonin gene-related peptide (CGRP)-positive nociceptive axons throughout dorsal horn and ventral horns. To restrict these axons within superficial laminas, adenovirus expressing semaphorin 3A was injected into the ventral spinal cord 3 d after NGF virus injection. Semaphorin 3A expression was observed in deep dorsal and ventral cord regions and limited axon growth to laminas I and II, shaping axonal regeneration toward the normal distribution pattern. NGF and semaphorin 3A treatment also targeted the regeneration of substance P-positive nociceptive axons but had no effect on injured isolectin B4-binding nociceptive axons. Axon regeneration led to functional restoration of nociception in both NGF- and NGF/semaphorin 3A-treated rats. Although no significant difference in behavior was found between these two groups, confocal microscopy illustrated ectopic synaptic formations in deeper laminas in NGF/green fluorescent protein-treated rats. The results suggested that antagonistic guidance cues can be used to induce and refine regeneration within the CNS, which is important for long-term, optimal functional recovery.