Cortical spreading depolarization (CSD) is a key pathophysiological event that underlies visual and sensory auras in migraine. CSD further drives the headache phase in migraine by promoting the activation and sensitization of trigeminal meningeal sensory afferents. The factors responsible for this meningeal nociceptive response in the wake of CSD remain poorly understood, but potentially involve the local release of algesic, proinflammatory mediators, including ATP. Here, we used an in vivo extracellular single-unit recording of meningeal afferent activity in combination with topical administration of pharmacological agents in a rat model to explore the role of ATP-P2X receptor signaling in mediating CSD-evoked meningeal afferent activation and sensitization. We found that broad-spectrum inhibition of P2X receptors, P2X7 receptor antagonist, and blockade of the related pannexin1 channel suppressed CSD-evoked afferent mechanical sensitization, but did not affect afferent activation. Pharmacological inhibition of the pronociceptive P2X2,3 ATP receptor did not affect meningeal nociception post-CSD. Finally, we provide evidence supporting the notion that the anti-nociceptive effect mediated by P2X7 is localized to the meningeal and not the cerebral cortex. We propose that meningeal P2X7 and Panx1 signaling, potentially in meningeal macrophages or neutrophils mediates the mechanical sensitization of meningeal afferents, and contributes to migraine pain by exacerbating the headache during normally innocuous physical activities.