Notch-1 (Notch) is a cell surface receptor that regulates cell-fate decisions in the developing nervous system and it may also have roles in synaptic plasticity in the adult brain. Binding of its ligands results in the proteolytic cleavage of Notch by the gamma-secretase enzyme complex, thereby causing the release of a Notch intracellular domain (NICD) that translocates to the nucleus, where it regulates transcription. Here we show that activation of Notch modulates ischemic neuronal cell death in vitro and in vivo. Specifically, our findings from the use of Notch-1 siRNA or the overexpression of NICD indicate that Notch activation contributes to cell death. Using modified NICD, we demonstrate an apoptosis-inducing function of NICD in both the nucleus and the cytosol. NICD transfection-induced cell death was reduced by blockade of calcium signaling, caspase activation and Janus kinase signaling. Inhibition of the Notch-activating enzyme, gamma-secretase, protected against ischemic neuronal cell death by targeting an apoptotic protease, cleaved caspase-3, nuclear factor kappa B (NFkappaB), and the prodeath BH3-only protein, Bim (Bcl-2-interacting mediator of cell death). Treatment of mice with a gamma-secretase inhibitor, compound E, reduced infarct size and improved functional outcome in a model of focal ischemic stroke. Furthermore, gamma-secretase inhibition reduced NICD, p-P65 and Bim levels in vivo. These findings suggest that Notch signaling endangers neurons following ischemic stroke by modulating the NFkappaB, prodeath protein Bim, and caspase pathways.