Calcineurin inhibitors, such as cyclosporin A and tacrolimus (FK506), have played a pivotal role in the preservation of allograft function. However, these drugs can cause unexplained severe pain in patients, often referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin can regulate NMDA receptor (NMDAR) activity, the causal relationship between spinal synaptic plasticity and CIPS remains unknown. In this study, we showed that systemic administration of FK506 (1.5 mg kg(-1) day(-1)) for 7 days in rats led to long-lasting nociceptive and mechanical hypersensitivity. Whole-cell patch-clamp recordings in spinal cord slices revealed that FK506 treatment caused a large increase in the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation. The amplitude of NMDAR currents elicited by puff NMDA application to dorsal horn neurons was also significantly greater in FK506-treated than in vehicle-treated rats. The frequency of spontaneous and miniature EPSCs in most dorsal horn neurons was profoundly increased in FK506-treated rats and was reduced by blocking NMDARs. Furthermore, blocking GluN2A or GluN2B subunits similarly reduced the amplitude of evoked EPSCs and the frequency of miniature EPSCs in dorsal horn neurons of FK506-treated rats. In addition, intrathecal injection of an NMDAR antagonist or systemic administration of memantine effectively reversed nociceptive and mechanical hypersensitivity in FK506-treated rats. Our findings indicate that calcineurin inhibition increases glutamate-mediated nociceptive input by potentiating presynaptic and postsynaptic NMDAR activity in spinal cords. NMDAR antagonists may represent a new therapeutic option for the treatment of CIPS.