Pannexin-1 (Panx1) is a large-pore ion and solute permeable channel highly expressed in the nervous system, where it subserves diverse processes, including neurite outgrowth, dendritic spine formation, and N-methyl D-aspartate (NMDA) receptor (NMDAR)-dependent plasticity. Moreover, Panx1 dysregulation contributes to neurological disorders, including neuropathic pain, epilepsy, and excitotoxicity. Despite progress in understanding physiological and pathological functions of Panx1, the mechanisms that regulate its activity, including its ion and solute permeability, remain poorly understood. In this study, we identify endoplasmic reticulum (ER)-resident stromal interaction molecules (STIM1/2), which are Ca²⁺ sensors that communicate events within the ER to plasma membrane channels, as binding and signaling partners of Panx1. We demonstrate that Panx1 is activated to its large-pore configuration in response to stimuli that recruit STIM1/2 and map the interaction interface to a hydrophobic region within the N terminus of Panx1. We further characterize a Panx1 N terminus–recognizing antibody as a function-blocking tool able to prevent large-pore Panx1 activation by STIM1/2. Using either the function-blocking antibody or re-expression of Panx1 deletion mutants in Panx1 knockout (KO) neurons, we show that STIM recruitment couples Ca²⁺ entry via NMDARs to Panx1 activation, thereby identifying a model of NMDAR-STIM-Panx1 signaling in neurons. Our study highlights a previously unrecognized and important role of the Panx1 N terminus in regulating channel activation and membrane localization. Considering past work demonstrating an intimate functional relation between NMDARs and Panx1, our study opens avenues for understanding activation modality and context-specific functions of Panx1, including functions linked to diverse STIM-regulated cellular responses.