To evaluate the optimal timing of mesenchymal stem cell (MSC) transplantation following stroke, rats were transplanted with MSCs at 1 (D1), 4 (D4), and 7 days (D7) after middle cerebral artery occlusion (MCAo). Rats in the D1 group showed a better functional recovery than those in the D4 or D7 groups after MCAo. MSCs preferentially migrated to the cortex in the D1 group, while the MSCs in the D4 or D7 groups preferentially migrated to the striatum. Interestingly, the level of monocyte chemotactic protein-1 (MCP-1) in the cortex was highest at 1 day after MCAo, while the level of stromal cell-derived factor-1 (SDF-1) in the striatum was lowest at 1 day after MCAo and then increased over time. The pattern of MCP-1 and SDF-1 level changes according to the time after MCAo was consistent with in vivo and in vitro migration patterns of MSCs. The results suggest that an earlier MSC transplantation is associated with a better functional recovery after stroke, which could be explained by the preferential migration of MSCs to the cortex in the early transplantation group. The time-dependent differential expression of MCP-1 and SDF-1 between ischemic regions seemed to mediate the differential migration of MSCs. Highest level of MCP-1 at one day of stroke may induce preferential migration of MSCs to the cortex, then better functional improvement.