The biological functions of matrix metalloproteinases (MMPs) extend beyond extracellular matrix degradation. Non‐proteolytic activities of MMPs are just beginning to be understood. Herein, we evaluated the role of proMMPs in cell migration. Employing a Transwell chamber migration assay, we demonstrated that transfection of COS‐1 cells with various proMMP cDNAs resulted in enhancement of cell migration. Latent MMP‐2 and MMP‐9 enhanced cell migration to a greater extent than latent MMP‐1, ‐3, ‐11 and ‐28. To examine if proteolytic activity is required for MMP‐enhanced cell migration, three experimental approaches, including fluorogenic substrate degradation assay, transfection of cells with catalytically inactive mutant MMP cDNAs, and addition of hydroxamic acid‐derived MMP inhibitors, were employed. We demonstrated that the proteolytic activities of MMPs are not required for MMP‐induced cell migration. To explore the mechanism underlying MMP‐enhanced cell migration, structure‐function relationship of MMP‐9 on cell migration was evaluated. By using a domain swapping approach, we demonstrated that the hemopexin domain of proMMP‐9 plays an important role in cell migration when examined by a transwell chamber assay and by a phagokinetic migration assay. TIMP‐1, which interacts with the hemopexin domain of proMMP‐9, inhibited cell migration, whereas TIMP‐2 had no effect. Employing small molecular inhibitors, MAPK and PI3K pathways were found to be involved in MMP‐9‐mediated cell migration. In conclusion, we demonstrated that MMPs utilize a non‐proteolytic mechanism to enhance epithelial cell migration. We propose that hemopexin homodimer formation is required for the full cell migratory function of proMMP‐9. J. Cell. Physiol. 217: 643–651, 2008. © 2008 Wiley‐Liss, Inc.