EphrinB3 may restrict axonal regeneration and recovery following traumatic injury of the adult mammalian central nervous system (CNS). Therefore, inhibition of ephrinB3 expression may enhance the ability of the nervous system to regenerate following damage. In the present study, lentiviral expressing vectors, pGCSIL-GFP, expressing an active small interfering RNA (siRNA) targeting the EphB3 sequence, were used to determine the effect of inhibiting EphB3 on nerve functional recovery and regeneration. Basso-Beattie-Bresnahan (BBB) locomotor scores and growth-associated protein, 43 kDa (GAP43) expression levels were determined at 1, 2, 4 and 8 weeks following transplantation of the siRNA. It was identified that EphB3 mRNA and protein levels in the siRNA group were significantly reduced (P<0.01) in the spine compared with the control group, at four weeks following intraparenchymal administration of the siRNA into the right lumbar. BBB locomotor scores were significantly increased (P<0.05) in siRNA animals compared with the sham group, at 1, 2, 4 and 8 weeks following transplantation of the siRNA. In addition, a significant increase in GAP43 expression was detected in the siRNA group compared with the control group. This preclinical study demonstrates that ephrinB3 silencing contributes to axonal growth regeneration and improves recovery from spinal cord injury (SCI).