Bone marrow-derived mononuclear cells (MNCs) enhance recovery in rodent stroke models. Since stroke activates the bone marrow, there may be biological differences of autologous MNCs derived poststroke compared with the prestroke setting. We analyzed MNCs harvested from the same Long Evans rats 1 day before and 1 day after ischemic stroke or sham stroke. Stroke was induced by suture occlusion of the middle cerebral artery for 90 min. MNCs were characterized by flow cytometry to identify differences in the percentages of different cell subpopulations. MNCs were also placed in culture and cytokines were measured in the media. In separate experiments, Long Evans rats received 24 h after stroke an intracarotid injection of saline or autologous MNCs, prepared from the same animal, either 1 day before or 1 day after stroke. The rats were then followed on the cylinder and corner tests for 28 days. In poststroke MNCs compared with prestroke MNCs, there was a significant reduction in T and mesenchymal stem cells and a significant increase in CD34+ and natural killer cells. Postsham MNCs showed an elevation in CD11b and CD45R cells compared with presham MNCs. The concentrations of IL-10, IL-6, MCP-1, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α were significantly increased in poststroke MNCs compared with prestroke MNCs. Postsham MNCs showed a decrease in VEGF. Poststroke MNCs in comparison with prestroke MNCs led to a greater recovery on neurological testing and reduced lesion size. Autologous MNCs exert different biological responses when derived from the poststroke setting compared with normal animals.