Objective: A study was undertaken to investigate the effect of neonatal hypoxic-ischemic (HI) brain damage and mesenchymal stem cell (MSC) treatment on the structure and contralesional connectivity of motor function-related cerebral areas. Methods: Brain remodeling after HI +/- MSC treatment in neonatal mice was analyzed using diffusion tensor magnetic resonance imaging, immunohistochemistry, anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluorescent pseudorabies virus (PRV). Results: MSC treatment after HI reduced contralesional rewiring taking place after HI. Following MSC treatment, fractional anisotropy values, which were increased in both ipsi- and contralesional cortices and decreased in the corpus callosum (CC) after HI, were normalized to the level observed in sham-operated mice. These results were corroborated by myelin basic protein intensity and staining pattern in these areas. Anterograde tracing of ipsilesional motor neurons showed that after MSC treatment, fewer BDA-positive fibers crossed the CC and extended into the contralesional motor cortex compared to HI mice. This remodeling was functional, because retrograde labeling showed increased connectivity between impaired (left) forepaw and the contralesional (left) motor cortex after HI, whereas MSC treatment reduced this connection and increased the connection between the impaired (left) forepaw and the ipsilesional (right) motor cortex. Finally, the extent of contralesional rewiring measured with BDA and PRV tracing was related to sensorimotor dysfunction. Interpretation: This is the first study to describe MSC treatment after neonatal HI markedly reducing contralesional axonal remodeling induced by HI brain injury. ANN NEUROL 2012;71:785796