The purpose of this study was to enhance our understanding of the mechanisms of neuronal death following focal cerebral ischemia and the neuroprotective effects of tamoxifen (TMX). The phosphorylation state of 31 protein kinases/signaling proteins and superoxide anion (O₂^-) production in the contralateral and ipsilateral cortex was measured following permanent MCAO (pMCAO) in ovariectomized rats treated with placebo or TMX. The study revealed that pMCAO modulated the phosphorylation of a number of kinases/proteins in the penumbra at 2h after pMCAO. Of significant interest, p-ERK1,2 was elevated significantly following pMCAO. TMX attenuated the elevation of pERK-1,2, an effect correlated with reduced infarct size. In situ detection of O₂^- production showed a significant elevation at 1–2h post-pMCAO in the ischemic cortex with enhanced oxidative damage detected at 24h. ERK activation may be downstream of free radicals, a suggestion supported by the findings that cells positive for O₂^- had high pERK activation, and that an SOD mimetic, tempol significantly attenuated pERK activation following MCAO. TMX treatment significantly reduced the MCAO-induced elevation of O₂^- production, oxidative damage and proapoptotic caspase-3 activation. Additionally, pMCAO induced a significant reduction in the levels of manganese superoxide dismutase (MnSOD), which scavenge O₂^-, an effect largely prevented by TMX treatment – thus providing a potential mechanistic basis for the antioxidant effects of TMX. As a whole, these studies suggest that TMX neuroprotection may be achieved via an antioxidant mechanism that involves enhancement of primarily MnSOD levels, with a corresponding reduction of O₂^- production, and downstream kinase and caspase-3 activation.