Cell migration is critically involved in inflammation, cancer, and development. In this study, transforming growth factor-β-induced protein (βig-h3) was identified as a substrate of matrix metalloproteinase-9 (MMP-9) by site-directed mutagenesis. βig-h3 has two cleavage sites with the consensus sequence Pro-Xaa-Xaa-Hy-(Ser/Thr) (Hy is a hydrophobic amino acid) (PGSFT beginning at amino acid 135 and PPMGT beginning at amino acid 501). Using recombinant human βig-h3 and MMP-9, βig-h3 from βig-h3-transfected HEK293F cells, and MMP-9 from MMP-9-transfected HEK293F cells, human macrophages, and neutrophils, we found that MMP-9 proteolytically cleaves βig-h3. Cleavage leads to the loss of its adhesive property and its release from extracellular matrix proteins, collagen IV, and fibronectin. Spheroids formed by increased cell-cell interactions were observed in βig-h3-transfected HEK293F cells but not in vehicle-transfected HEK293F cells. In human glioma U87MG cells, MMP-9 constitutive overexpression resulted in endogenous βig-h3 cleavage. βig-h3 cleavage by MMP-9 led to increased cell invasion, and βig-h3 knockdown also resulted in increased cell invasion. The βig-h3 fragment cleaved by MMP-9 could bind to the surface of macrophages, and it may play a role as a peptide chemoattractant by inducing macrophage migration via focal adhesion kinase/Src-mediated signal activation. Thus, intact βig-h3 is responsible for cell migration inhibition, cell-cell contact, and cell-extracellular matrix interaction. Experimental evidence indicates that MMP-9-cleaved βig-h3 plays a role in MMP-9-mediated tumor cell and macrophage migration.