Background: Previously we proved that intracerebroventricular transplatation of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) significantly attenuated post hemorrhagic ventricular dilatation (PHVD) and brain injury, and improved behavior function in the newborn rats with severe intraventricular hemorrhage (IVH). Objective: We investigated whether knockdown of brain-derived neurotrophic factor (BDNF) by siRNA reduces the effectiveness of hUCB-MSCs on the IVH in the newborn rat. Methods BDNF siRNA was made with Lipofectamine RNAiMAX (Invitrogen) transfection. In vitro analysis demonstrated 20% at 48h of BDNF secretion after interference with BDNF signaling in MSCs compared to MSCs with scramble siRNA. Severe IVH was induced by intracerebroventricular injection of mother\'s rat blood (200ul) at postnatal day (P) 4. After confirming severe IVH by MRI at P5, 5x105 cells (MSC, MSC with scramble siRNA, or MSC with BNDF siRNA) were administered intracerebroventricular at postnatal day (P) 6 (IM, IsC, and IsBDNF groups). Follow-up brain MRI and behavior function tests including rotarod test and negative geotaxis test were done before harvest of brain tissue at P32 for morphological analysis. Results: Progressive hydrocephalus evidenced by increased ratio of ventricle/whole brain volume on the volumetric analysis of MRI was significantly attenuated in the IM and IsC groups, but not IsBDNF group, compared to IVH control group (p＜.05). Impaired behavior function induced from severe IVH, was significantly improved by in the IM and IsC groups (p＜.05), but not IsBDNF group. In morphometric analysis, IVH induced increased cell death, augmented reactive gliosis, decreased myelination were significantly improved by in the IM, IsC, and IsBDNF groups (p＜.05), however IsBDNF group showed less improvement than IM or IsC. Conclusions: We suggest that BDNF is an essential mediator in the brain protective action of hUCB-MSCs on the neonatal IVH in the newborn rats.