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.