Thrombin and iron are two major players in intracerebral hemorrhage-induced brain injury and our recent study found that thrombin contributes to hydrocephalus development in a rat model of intraventricular hemorrhage (IVH). This study investigated the role of red blood cell (RBC) lysis and iron in hydrocephalus after IVH. There were three parts to this study. First, male Sprague-Dawley rats received an injection of saline, packed, or lysed RBCs into the right lateral ventricle. Second, rats had an intraventricular injection of iron or saline. Third, the rats received intraventricular injection of lysed RBCs mixed with deferoxamine (0.5 mg in 5 μL saline) or saline. All rats underwent magnetic resonance imaging at 24 hours and were then euthanized for brain edema measurement, western blot analysis, or brain histology. We found that intraventricular injection of lysed RBCs, but not packed RBCs, resulted in ventricular enlargement and marked increases in brain heme oxygenase-1 and ferritin at 24 hours. Intraventricular injection of iron also resulted in ventricular enlargement and ventricular wall damage 24 hours later. Coinjection of deferoxamine reduced lysed RBC-induced ventricular enlargement (P<0.01). These results suggest that iron, a degradation product of hemoglobin, has an important role in hydrocephalus development after IVH.