3-O-caffeoylquinic acid (3-CQA) is an isomer of chlorogenic acid, which has been shown to regulate lipopolysaccharide-induced tumor necrosis factor production in microglia. Whereas overactivation of microglia is associated with neuronal loss in brain diseases via reactive oxygen species (ROS) production and glutamate excitotoxicity, naive (nonactivated) microglia are believed to generate little ROS under basal conditions, contributing to the modulation of synaptic activity and nerve tissue repair. However, the signaling pathways controlling basal ROS homeostasis in microglial cells are still poorly understood. Here we used time-lapse microscopy coupled with highly sensitive FRET biosensors (for detecting c-Src activation, ROS generation, and glutamate release) and lentivirus-mediated shRNA delivery to study the pathways involved in antioxidant-regulated ROS generation and how this associates with microglia-induced neuronal cell death. We report that 3-CQA abrogates the acquisition of an amoeboid morphology in microglia triggered by Abeta oligomers or the HIV Tat peptide. Moreover, 3-CQA deactivates c-Src tyrosine kinase and abrogates c-Src activation during proinflammatory microglia stimulation, which shuts off ROS production in these cells. Moreover, forced increment of c-Src catalytic activity by overexpressing an inducible c-Src heteromerization construct in microglia increases ROS production, abrogating the 3-CQA effects. Whereas oxidant (hydrogen peroxide) stimulation dramatically enhances glutamate release from microglia, such release is diminished by the 3-CQA inhibition of c-Src/ROS generation, significantly alleviating cell death in cultures from embryonic neurons. Overall, we provide further mechanistic insight into the modulation of ROS production in cortical microglia, indicating antioxidant-regulated c-Src function as a pathway for controlling microglia-triggered oxidative damage.