12/15-lipoxygenase (12/15-LOX) contributes to acute neuronal injury and edema formation in mouse models of middle cerebral artery occlusion (MCAO). The apoptosis-inducing factor (AIF) is implicated in caspase-independent forms of apoptosis, and has been linked to ischemic neuronal cell death. We show here that increased AIF in the peri-ischemic cortex of mouse colocalizes with 12/15-LOX after 2 h of MCAO. The 12/15-LOX inhibitor baicalein prevents the increase and nuclear localization of AIF, suggesting this pathway may be partially responsible for the neuroprotective qualities of baicalein. Using an established cell line model of neuronal oxidative stress, we show that 12/15-LOX activated after glutathione depletion leads to AIF translocation to the nucleus, which is abrogated by the 12/15-LOX inhibitor baicalein (control: 19.3%+/-6.8% versus Glutamate: 64.0%+/-8.2% versus glutamate plus baicalein: 11.4%+/-2.2%). Concomitantly, resident proteins of the ER are dispersed throughout the cell (control: 31.0%+/-8.4% versus glutamate: 70.0%+/-5.5% versus glutamate plus baicalein: 8.0%+/-2.7%), suggesting cell death through organelle damage. Taken together, these findings show that 12/15-LOX and AIF are sequential actors in a common cell death pathway that may contribute to stroke-induced brain damage.