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It has recently been revealed that during the aorta-clamped period, D-Ala2, D-Leu5-Enkephalin (DADLE) infusion can protect the spinal cord against ischemia and reperfusion (I/R) injury. However, the protective effects of DADLE administration prior to ischemia or at the time of early reperfusion have not yet been investigated. Drug pre- or post-conditioning can serve as a more valuable clinical strategy. Therefore, the present study was designed to investigate the neuroprotective effect of DADLE infusion at different time intervals in order to determine the optimum time point for ischemic spinal cord protection. A total of 40 New Zealand white rabbits were randomly divided into 5 groups: Sham-operated (Sham), normal saline pre-conditioning (NS), DADLE per-conditioning (Dper), DADLE pre-conditioning (Dpre) and DADLE post-conditioning (Dpost). All animals were subjected to spinal cord ischemia for 30 min followed by 48 h reperfusion. Hind limb motor functions were assessed according to the Tarlov criterion when the animals regained consciousness, 6, 24 and 48 h after reperfusion. Histological analysis and the number of viable α-motor neurons were also used to assess the extent of spinal cord injury. Compared with the NS group, the Tarlov scores and the number of normal neurons were significantly higher in the Dper group (P