The pannexin ion channels are members of the gap junction superfamily. Unlike their connexin cousins, pannexins function as bona fide ion channels in the plasma membrane, and do not likely form intercellular junctions. Pannexin-1 (Panx1) is activated during ischaemia, contributing to the anoxic depolarization (Thompson et al., 2008) and suggesting that Panx1 may be important for cell death. The mechanism of Panx1 activation involves the N-methyl-D-aspartate receptor (NMDAR) and Src family kinases (SFKs) (Weilinger et al., 2012). Here we tested the hypothesis that NMDAR-SFK-Panx1 signalling induces neuronal death during ischaemia through mitochondrial dysfunction. We used patch-clamp recording and live cell imaging from hippocampal brain slices / neuronal cultures prepared from isofluorane (inhaled) anesthetised rats or mice to determine if there is a direct link between Panx1 and mitochondrial function. Anoxia opened Panx1 channels because Panx1 antagonists or knockout substantially reduced the anoxic depolarization. Block of the NMDAR with APV also reduced the anoxic depolarization and the combined effect of APV and Panx1 blockers was not summative. We designed a small interfering peptide, Panx305-318 that mimics the SFK consensus site on the Panx1 c-terminal. Panx305-318 potently blocked ischaemia-induced Panx1 opening in brain slices and cultured neurons. Interestingly, Panx1, and not NMDARs, was critical for ischaemia-induced Ca2+-dysregulation and neuronal death. Panx1 block with Panx305-318 attenuated mitochondrial depolarization and permeability transition pore opening, measured by live cell fluorescent imaging. Taken together, these data suggest that Ca2+ influx through NMDAR activated Panx1 channels is specifically coupled to mitochondrial dysfunction and neuronal death during ischaemia.