Backgrounds Previously we proved that transplatation of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) significantly attenuated brain injury in the newborn rats with severe IVH. The aim of this study was to optimize the timing of MSCs transplantation to prevent PHVD and ensuing brain damage after severe IVH. Methods 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, hUCB-MSCs (1x105) were transplanted intracerebroventriculary at P6 (early group) or P11 (late group). 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 morphometric analysis. Result PHVD, which evidenced by increased ratio of ventricle/whole brain volume on the volumetric analysis of MRI, was significantly attenuated in the early MSCs transplantation group (p-value0.05), but not in late group. Impaired behavior function induced from severe IVH, was significantly improved by early MSCs transplantation at P6 (p-value0.05), but not by late MSCs injection at P11. In morphometric analysis, reduced corpus callosum thickness, increased cell death, and augmented reactive gliosis were improved in early MSCs transplantation group (p-value0.05), but not in late group. Conclusion Intracerebroventricular transplatation of hUCB-MSCs time-dependently attenuated PHVD and brain injury, and improved behavior function in the newborn rats with severe IVH, showing significant protection only in the early but not in the late phase of brain damage after severe IVH.