Human mesenchymal stem cells (MSCs) from bone marrow stroma can home to and repair injured tissue, but the rate of engraftment is generally low. Regulating migration-related signaling of MSCs may be a powerful strategy to enhance this process. To gain insight into the molecular mechanisms governing homing, we identified negative factors affecting MSC migration using an in vitro model of injured lung. Heat-labile factors in bovine pituitary extract, a component of serum-free epithelial medium, inhibited more than 97% of MSC migration. This was partly due to a dose-dependent response to macrophage migration inhibitory factor (MIF). Eighty-five ng/mL recombinant MIF, the concentration found in the epithelial medium, inhibited about 50% of MSC migration. Media conditioning by uninjured or bleomycin-injured bronchial epithelial cells partially attenuated this suppressive effect. Additionally, the anti-inflammatory agent ISO-1, a small-molecule MIF antagonist, further increased MSC migration by nearly fourfold in conditioned epithelial media. This is the first report of the effect of MIF and ISO-1 on MSC migration, and the data suggest that MIF and its antagonists may have therapeutic applications in controlling MSC homing during repair of injured lung and in other clinically relevant systems.