Although ex vivo culture expansion is necessary to use autologous mesenchymal stem cells (MSCs) in treating stroke patients, and several researchers have utilized culture-expanded cells in their studies, the effects of culture expansion on neurogenesis and trophic support are unknown. Thus, we evaluated the impact of the passage of MSCs on their effects in a rat stroke model. The i.v. application of ex vivo-cultured human MSCs, earlier (passage 2) or later passage (passage 6), was performed in a rat stroke model. Behavioral tests, immunohistochemical studies, and quantitative analysis using the CAST-grid system were performed to evaluate the degree of neurogenesis. We also evaluated the levels of trophic factors in both control and MSC-treated rat brain extract. Compared to rats that received later-passage human MSCs, behavioral recovery and neurogenesis as revealed by bromodeoxyuridine staining were more pronounced in rats that received earlier-passage human MSCs (p < 0.01 in both cases). Double staining showed that most of the endogenous neuronal progenitor cells, but few human MSCs, expressed neuronal and glial phenotypes. Tissue levels of trophic factors, including glial cell line-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, and hepatocyte growth factor, were higher in earlier-passage MSC-treated brains than in control or later-passage MSC-treated brains (p < 0.01 in all cases). Our results indicate that ischemia-induced neurogenesis was enhanced by the i.v. administration of human MSCs. The effects were more pronounced with earlier-passage than with later-passage human MSCs, which may be related to the differential capacity in trophic support, depending on their passage.