The metalloprotease ADAMTS7, a drug target in atherosclerosis and vascular restenosis, is thought to have no relevant physiological role in the cardiovascular system. However, potential cooperation with its close homolog, ADAMTS12, that may mask such a role remains unexplored. The objective of the present work was to investigate cardiac biology in mice with genetic inactivation of both proteases and to define their proteolytic activities in a relevant matrisome. Here, we demonstrate that these genes are co-expressed in heart valves and buffer the loss of each other by compensatory upregulation. Doppler echocardiography showed that adult Adamts7-/-;Adamts12-/- mice had significant regurgitant aortic valves. Leaflets of Adamts7-/-;Adamts12-/- aortic valves, but not the respective single mutants, were already abnormally shaped at birth and subsequently underwent severe disorganization and enlargement postnatally with calcification near the valve annuli. We further comprehensively identified novel ADAMTS7 and ADAMTS12 substrates relevant to the valve matrisome in secretome libraries from Adamts7-/-;Adamts12-/- cells using the N-terminomics technique Terminal Amine Isotopic Labeling of Substrates (TAILS). Although ADAMTS7 and ADAMTS12 shared several extracellular matrix (ECM) substrates, the cleavage sites and sequence preferences for each protease were distinct. Adamts7-/-;Adamts12-/- valve leaflets showed accumulation of several of the identified ECM substrates tested, including periostin, a matricellular protein crucial for cardiac valve homeostasis. We conclude that the observed myxomatous heart valve degeneration reflects accumulation of diverse ADAMTS7 and ADAMTS12 ECM substrates, as well as perturbation of regulatory pathways with roots in ECM, such as TGFsignaling, which was increased in Adamts7-/-;Adamts12-/- valve leaflets, and propagates ECM dysregulation. These findings are highly relevant to current interest in drugs targeting ADAMTS7 for applications such as mitigation of atherosclerosis or vascular re-stenosis.