Whereas iron chelators were shown to induce neuroprotection against brain injury, the effect of iron chelators on ischemia-induced damage of cerebral endothelium is largely unknown. Our objective was to explore the endothelioprotective effect of the lipophilic iron chelator dipyridyl (DP) (i) in vitro on the death of cerebral endothelial cells (CECs) subjected to intracellular iron loading and (ii) in vivo on the ischemia-induced blood-brain barrier (BBB) disruption. When given shortly after iron exposure or brain ischemia, DP prevented the death of CECs and diminished BBB disruption, respectively, whereas a delayed administration of DP was associated with a lower CECs protection. Interestingly, when given preventively, DP also abrogated the death of CECs and reduced BBB disruption. However, a long delay between DP treatment and iron exposure led to a higher protection, suggesting a preconditioning effect of DP. Accordingly, prevention of hydroxyl radical formation through iron chelation cannot explain alone the beneficial effect of preventive DP treatment. Our findings showing that DP failed to induce the potentially cytoprotective proteins, heme oxygenase-1 and manganese superoxide dismutase, suggest that other proteins participate to the preconditioning effect of DP. To conclude, the curative and preventive effects of DP evidenced in this study suggest that iron chelation therapy represents a favorable and effective approach to increase BBB resistance towards ischemic injury.