Pannexin-1 (Px1) is expressed at postsynaptic sites in pyramidal neurons, suggesting that these hemichannels contribute to dendritic signals associated with synaptic function. We found that, in pyramidal neurons, N-methyl-d-aspartate receptor (NMDAR) activation induced a secondary prolonged current and dye flux that were blocked with a specific inhibitory peptide against Px1 hemichannels; knockdown of Px1 by RNA interference blocked the current in cultured neurons. Enhancing endogenous NMDAR activation in brain slices by removing external magnesium ions (Mg2+) triggered epileptiform activity, which had decreased spike amplitude and prolonged interburst interval during application of the Px1 hemichannel blocking peptide. We conclude that Px1 hemichannel opening is triggered by NMDAR stimulation and can contribute to epileptiform seizure activity.