Extracellular matrix protein 1 (ECM1), an approximately 85-kDa glycoprotein with broad tissue distribution, harbors mutations in lipoid proteinosis (LP), a heritable disease characterized by reduplication of basement membranes and hyalinization of dermis, associated with neurologic disorders. The mechanisms leading from ECM1 mutations to LP phenotype are unknown. In this study, we explored ECM1 protein-protein interactions utilizing yeast two-hybrid genetic screen of human placental library, which identified nine interacting proteins, including matrix metalloproteinase 9 (MMP9). The interactions were confirmed by beta-galactosidase assay with isolated clones and by co-immunoprecipitation which narrowed the interacting segment in ECM1 to the C-terminal tandem repeat 2 (amino acids 236-361). This peptide segment also inhibited MMP9 activity in a gelatin-based ELISA assay. We propose that ECM1-mediated reduction in MMP9 proteolytic activity may have relevance to pathogenesis of LP.