Recent findings suggest that Notch-1 signaling contributes to neuronal death in ischemic stroke, but the underlying mechanisms are unknown. Hypoxia inducible factor-1alpha (HIF-1alpha), a global regulator of cellular responses to hypoxia, can interact with Notch and modulate its signaling during hypoxic stress. Here we show that Notch signaling interacts with the HIF-1alpha pathway in the process of ischemic neuronal death. We found that a chemical inhibitor of the Notch-activating enzyme, gamma-secretase, and a HIF-1alpha inhibitor, protect cultured cortical neurons against ischemic stress, and combined inhibition of Notch-1 and HIF-1alpha further decreased neuronal death. HIF-1alpha and Notch intracellular domain (NICD) are co-expressed in the neuronal nucleus, and co-immunoprecipitated in cultured neurons and in brain tissue from mice subjected to focal ischemic stroke. Overexpression of NICD and HIF-1alpha in cultured human neural cells enhanced cell death under ischemia-like conditions, and a HIF-1alpha inhibitor rescued the cells. RNA interference-mediated depletion of endogenous NICD and HIF-1alpha also decreased cell death under ischemia-like conditions. Finally, mice treated with inhibitors of gamma-secretase and HIF-1alpha exhibited improved outcome after focal ischemic stroke, with combined treatment being superior to individual treatments. Additional findings suggest that the NICD and HIF-1alpha collaborate to engage pro-inflammatory and apoptotic signaling pathways in stroke.