Oxidized glutathione (GSSG) is commonly viewed as a byproduct of GSH metabolism. The pathophysiological significance of GSSG per se remains poorly understood. Adopting a microinjection approach to isolate GSSG elevation within the cell, this work identifies that GSSG can trigger neural HT4 cell death via a 12-lipoxygenase (12-Lox)-dependent mechanism. In vivo, stereotaxic injection of GSSG into the brain caused lesion in wild-type mice but less so in 12-Lox knockout mice. Microinjection of graded amounts identified 0.5 mM as the lethal [GSSG]i in resting cells. Interestingly, this threshold was shifted to the left by 20-fold (0.025 mM) in GSH-deficient cells. This is important because tissue GSH lowering is commonly noted in the context of several diseases as well as in aging. Inhibition of GSSG reductase by BCNU is known to result in GSSG accumulation and caused cell death in a 12-Lox-sensitive manner. GSSG S-glutathionylated purified 12-Lox as well as in a model of glutamate-induced HT4 cell death in vitro where V5-tagged 12-Lox was expressed in cells. Countering glutamate-induced 12-Lox S-glutathionylation by glutaredoxin-1 overexpression protected against cell death. Strategies directed at improving or arresting cellular GSSG clearance may be effective in minimizing oxidative stress-related tissue injury or potentiating the killing of tumor cells, respectively.