Morphine loses analgesic potency after repeated administration. The underlying mechanism is not fully understood. Glia are thought to be involved in morphine tolerance, and P2X7 purinergic receptor (P2X7R) has been implicated in neuron-glia communication and chronic pain. The present study demonstrated that P2X7R immunoreactivity was colocalized with the microglial marker OX42, but not the astrocytic marker GFAP, in the spinal cord. The protein level of spinal P2X7R was upregulated after chronic exposure to morphine. Intrathecal administration of Brilliant Blue G (BBG), a selective P2X7R inhibitor, significantly attenuated the loss of morphine analgesic potency, P2X7R upregulation, and microglial activation. Furthermore, RNA interference targeting the spinal P2X7R exhibited a similar tolerance-attenuating effect. Once morphine analgesic tolerance is established, it was no longer affected by intrathecal BBG. Together, our results suggest that spinal P2X7R is involved in the induction but not maintenance of morphine tolerance. Copyright © 2010 the authors.