Anoxic depolarization of pyramidal neurons results from a large inward current that is activated, in part, by excessive glutamate release during exposure to anoxia/ischemia. Pannexin-1 (Panx1) channels can be activated both by ischemia and NMDA receptors (NMDARs), but the mechanisms of Panx1 activation are unknown. We used whole-cell recordings to show that pharmacological inhibition or conditional genetic deletion of Panx1 strongly attenuates the anoxic depolarization of CA1 pyramidal neurons in acute brain slices from rats and mice. Anoxia or exogenous NMDA activated Src family kinases (SFKs), as measured by increased phosphorylation of SFKs at Y416. The SFK inhibitor PP2 prevented Src activation and Panx1 opening during anoxia. A newly developed interfering peptide that targets the SFK consensus-like sequence of Panx1 (Y308) attenuated the anoxic depolarization (AD) without affecting SFK activation. Importantly, the NMDAR antagonists,d-APV and R-CPP, attenuated AD currents carried by Panx1, and the combined application ofd-APV and10panx (a Panx1 blocker) inhibited AD currents to the same extent as either blocker alone. We conclude that activation of NMDARs during anoxia/ischemia recruits SFKs to open Panx1, leading to sustained neuronal depolarizations.