Hypoxia causes oxidative stress and excitotoxicity, culminating in neuronal damage during brain ischemia. Hypoxia also activates microglia, the myeloid resident cells of the brain parenchyma. Upon activation, microglia release high amounts of the neurotransmitter glutamate, contributing for neuronal excitotoxicity during brain insults. Here, we reveal a signaling pathway controlling glutamate release from human microglia during hypoxia. We show that hypoxia-mediated redox imbalance promotes the activation of endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP3) receptors leading to Ca(2+) mobilization into the cytosol. Increasing cytosolic Ca(2+) signaling in microglia activates the non-receptor protein tyrosine kinase Src at the plasma membrane. Src activation enhances the permeability of microglial gap junctions promoting the release of glutamate during hypoxia. Preventing the hypoxia-triggered redox imbalance, using the dietary antioxidants neochlorogenic acid or vitamin C, inhibits InsP3-dependent Ca(2+) signaling and abrogates the release of glutamate. Overall, modulating microglial Ca(2+) signaling in response to changes in the redox microenvironment might be critical for controlling glutamate excitotoxicity during hypoxia.