Mesenchymal stem cells (MSCs) reside within tissues such as bone marrow, cord blood, and dental pulp and can differentiate into other mesenchymal cell types. Differentiated MSCs, called circulating fibrocytes, have been demonstrated in human lungs and migrate to injured lung tissue in experimental models. It is likely that MSCs migrate from the bone marrow to sites of injury by following increasing chemokine concentrations. In the present study, we show that primary mouse bone marrow mesenchymal stem cells (BM-MSCs) exhibit directed chemotaxis through transwell inserts toward increasing concentrations of the chemokines complement component 5a, stromal cell-derived factor-1alpha, and monocyte chemotactic protein-1. Prior research has indicated that myristoylated alanine-rich C kinase substrate (MARCKS) protein is critically important for motility in macrophages, neutrophils, and fibroblasts, and here we investigated a possible role for MARCKS in BM-MSC directed chemotaxis. The presence of MARCKS in these cells as well as in human cord blood MSC was verified by Western blotting, and MARCKS was rapidly phosphorylated in these cells after exposure to chemokines. A synthetic peptide that inhibits MARCKS function attenuated, in a concentration-dependent manner, directed chemotaxis of BM-MSCs, while a missense control peptide had no effect. Our results illustrate, for the first time, that MARCKS protein plays an integral role in BM-MSC-directed chemotaxis in vitro.