Non-proteolytic activities of matrix metalloproteinases (MMPs) have recently been shown to impact cell migration, but the precise mechanism remains to be understood. We previously demonstrated that the hemopexin (PEX) domain of MMP-9 is a prerequisite for enhanced cell migration. Using a biochemical approach, we now report that dimerization of MMP-9 through the PEX domain appears necessary for MMP-9-enhanced cell migration. Following a series of substitution mutations within the MMP-9 PEX domain, blade IV was shown to be critical for homodimerization, whereas blade I was required for heterodimerization with CD44. Blade I and IV mutants showed diminished enhancement of cell migration compared with wild type MMP-9-transfected cells. Peptides mimicking motifs in the outermost strands of the first and fourth blades of the MMP-9 PEX domain were designed. These peptides efficiently blocked MMP-9 dimer formation and inhibited motility of COS-1 cells overexpressing MMP-9, HT-1080, and MDA-MB-435 cells. Using a shRNA approach, CD44 was found to be a critical molecule in MMP-9-mediated cell migration. Furthermore, an axis involving a MMP-9-CD44-EGFR signaling pathway in cell migration was identified using antibody array and specific receptor tyrosine kinase inhibitors. In conclusion, we dissected the mechanism of pro-MMP-9-enhanced cell migration and developed structure-based inhibitory peptides targeting MMP-9-mediated cell migration.