Glutathione depletion and 12-lipoxygenase-dependent metabolism of arachidonic acid are known to be implicated in neurodegeneration associated with acute ischemic stroke. The objective of this study was to investigate the significance of miR-29 in neurodegeneration associated with acute ischemic stroke. Neural cell death caused by arachidonic acid insult of glutathione-deficient cells was preceded by a 12-lipoxygenase-dependent loss of miR-29b. Delivery of miR-29b mimic to blunt such loss was neuroprotective. miR-29b inhibition potentiated such neural cell death. 12-Lipoxygenase knockdown and inhibitors attenuated the loss of miR-29b in challenged cells. In vivo, stroke caused by middle-cerebral artery occlusion was followed by higher 12-lipoxygenase activity and loss of miR-29b as detected in laser-captured infarct site tissue. 12-Lipoxygenase knockout mice demonstrated protection against such miR loss. miR-29b gene delivery markedly attenuated stroke-induced brain lesion. Oral supplementation of α-tocotrienol, a vitamin E 12-lipoxygenase inhibitor, rescued stroke-induced loss of miR-29b and minimized lesion size. This work provides the first evidence demonstrating that loss of miR-29b at the infarct site is a key contributor to stroke lesion. Such loss is contributed by activity of the 12-lipoxygenase pathway providing maiden evidence linking arachidonic acid metabolism to miR-dependent mechanisms in stroke.