AIMS: Cardiac fibrosis is a major cause of heart failure (HF), and mediated by the differentiation of cardiac fibroblasts into myofibroblasts. However, limited tools are available to block cardiac fibrosis. ADAMTS16 is a member of the ADAMTS superfamily of extracellular protease enzymes involved in extracellular matrix (ECM) degradation and remodelling. In this study, we aimed to establish ADAMTS16 as a key regulator of cardiac fibrosis. METHODS AND RESULTS: Western blot and qRT-PCR analyses demonstrated that ADAMTS16 was significantly up-regulated in mice with transverse aortic constriction (TAC) associated with left ventricular hypertrophy and HF, which was correlated with increased expression of Mmp2, Mmp9, Col1a1, and Col3a1. Overexpression of ADAMTS16 accelerated the AngII-induced activation of cardiac fibroblasts into myofibroblasts. Protein structural analysis and co-immunoprecipitation revealed that ADAMTS16 interacted with the latency-associated peptide (LAP)-transforming growth factor (TGF)-β via a RRFR motif. Overexpression of ADAMTS16 induced the activation of TGF-β in cardiac fibroblasts; however, the effects were blocked by a mutation of the RRFR motif to IIFI, knockdown of Adamts16 expression, or a TGF-β-neutralizing antibody (ΝAb). The RRFR tetrapeptide, but not control IIFI peptide, blocked the interaction between ADAMTS16 and LAP-TGF-β, and accelerated the activation of TGF-β in cardiac fibroblasts. In TAC mice, the RRFR tetrapeptide aggravated cardiac fibrosis and hypertrophy by up-regulation of ECM proteins, activation of TGF-β, and increased SMAD2/SMAD3 signalling, however, the effects were blocked by TGF-β-NAb. CONCLUSION: ADAMTS16 promotes cardiac fibrosis, cardiac hypertrophy, and HF by facilitating cardiac fibroblasts activation via interacting with and activating LAP-TGF-β signalling. The RRFR motif of ADAMTS16 disrupts the interaction between ADAMTS16 and LAP-TGF-β, activates TGF-β, and aggravated cardiac fibrosis and hypertrophy. This study identifies a novel regulator of TGF-β signalling and cardiac fibrosis, and provides a new target for the development of therapeutic treatment of cardiac fibrosis and HF.