Tolerance to the analgesic effect of an opioid is a pharmacological phenomenon that occurs after its prolonged administration. Activation of the NMDA receptor (NMDAR) has been implicated in the cellular mechanisms of opioid tolerance. However, activation of NMDARs can lead to neurotoxicity under many circumstances. Here we demonstrate that spinal neuronal apoptosis was induced in rats made tolerant to morphine administered through intrathecal boluses or continuous infusion. The apoptotic cells were predominantly located in the superficial spinal cord dorsal horn, and most apoptotic cells also expressed glutamic acid decarboxylase, a key enzyme for the synthesis of the inhibitory neurotransmitter GABA. Consistently, increased nociceptive sensitivity to heat stimulation was observed in these same rats. Mechanistically, the spinal glutamatergic activity modulated morphine-induced neuronal apoptosis, because pharmacological perturbation of the spinal glutamate transporter activity or coadministration of morphine with the NMDAR antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate affected both morphine tolerance and neuronal apoptosis. At the intracellular level, prolonged morphine administration resulted in an upregulation of the proapoptotic caspase-3 and Bax proteins but a downregulation of the antiapoptotic Bcl-2 protein in the spinal cord dorsal horn. Furthermore, coadministration with morphine of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (a pan-caspase inhibitor) or acetyl-aspartyl-glutamyl-valyl-aspart-1-aldehyde (a relatively selective caspase-3 inhibitor) blocked morphine-induced neuronal apoptosis. Blockade of the spinal caspase-like activity also partially prevented morphine tolerance and the associated increase in nociceptive sensitivity. These results indicate an opioid-induced neurotoxic consequence regulated by the NMDAR-caspase pathway, a mechanism that may have clinical implications in opioid therapy and substance abuse.