Cerebrovascular inflammation contributes to secondary brain injury following ischemia. Recent in vitro studies of cell migration and molecular guidance mechanisms have indicated that the Slit family of secreted proteins can exert repellant effects on leukocyte recruitment in response to chemoattractants. Utilizing intravital microscopy, we addressed the role of Slit in modulating leukocyte dynamics in the mouse cortical venular microcirculation in vivo following TNFα application or global cerebral ischemia. We also studied whether Slit affected neuronal survival in the mouse global ischemia model as well as in mixed neuronal-glial cultures subjected to oxygen-glucose deprivation. We found that systemically administered Slit significantly attenuated cerebral microvessel leukocyte-endothelial adherence occurring 4 h after TNFα and 24 h after global cerebral ischemia. Administration of RoboN, the soluble receptor for Slit, exacerbated the acute chemotactic response to TNFα. These findings are indicative of a tonic repellant effect of endogenous Slit in brain under acute proinflammatory conditions. Three days of continuous systemic administration of Slit following global ischemia significantly attenuated the delayed neuronal death of hippocampal CA1 pyramidal cells. Moreover, Slit abrogated neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation. The ability of Slit to reduce the recruitment of immune cells to ischemic brain and to provide cytoprotective effects suggests that this protein may serve as a novel anti-inflammatory and neuroprotective target for stroke therapy.