Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24-204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nM and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease.