ATP can activate a variety of pathways through P2 purinoreceptors, leading to neuroprotection and pathology in the CNS. Among all P2X receptors, the P2X7 receptor (P2X7R) is a well-defined therapeutic target for inflammatory and neuropathic pain. Activation of P2X7R can generate reactive oxygen species (ROS) in macrophages and microglia. However, the role of ROS in P2X7R-induced pain remains unexplored. Here, we investigated the downstream effects of neuronal P2X7R activation in the spinal cord. We found that ATP induces ROS production in spinal cord dorsal horn neurons, an effect eliminated by ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) and P2X7R antagonist A438079. A similar effect was observed with a P2X7R agonist, BzATP, and was attenuated by a NADPH oxidase inhibitor apocynin. Intrathecal administration of BzATP resulted in ROS production in the spinal cord and oxidative DNA damage in dorsal horn neurons. BzATP also induced robust biphasic spontaneous nociceptive behavior. Pre-treatment with A438079 abolished all BzATP-induced nociceptive behaviors, while ROS scavengers dose-dependently attenuated the secondary response. Here, we provide evidence that neuronal P2X7R activation leads to ROS production and subsequent nociceptive pain in mice. Together, the data indicate that P2X7R-induced ROS play a critical role in the P2X7R signaling pathway of the CNS.