AbstractBackground: Trabeculation is an integral component of cardiac ventricular morphogenesis and is dependent on the matrix metalloproteinase, ADAMTS1. A substrate of ADAMTS1 is the proteoglycan versican which is expressed in the developing ventricle and which has been implicated in trabeculation. Fibulin‐1 is a versican and ADAMTS1‐binding extracellular matrix protein required for ventricular morphogenesis. Here we investigated the involvement of fibulin‐1 in ADAMTS1‐mediated cleavage of versican in vitro, and the involvement of fibulin‐1 in versican cleavage in ventricular morphogenesis. Results: We show that fibulin‐1 is a cofactor for ADAMTS1‐dependent in vitro cleavage of versican V1, yielding a 70‐kDa amino‐terminal fragment. Furthermore, fibulin‐1‐deficiency in mice was found to cause a significant reduction (>90%) in ventricular levels of the 70‐kDa versican V1 cleavage product and a 2‐fold increase in trabecular cardiomyocyte proliferation. Decreased versican V1 cleavage and augmented trabecular cardiomyocyte proliferation in fibulin‐1 null hearts is accompanied by increased ventricular activation of ErbB2 and Erk1/2. By contrast, versican deficiency was found to lead to decreased cardiomyocyte proliferation and reduced ventricular trabeculation. Conclusion: We conclude that fibulin‐1 regulates versican‐dependent events in ventricular morphogenesis by promoting ADAMTS1 cleavage of versican leading to suppression of trabecular cardiomyocyte proliferation mediated by the ErbB2‐Map kinase pathway. Developmental Dynamics 241:303–314, 2012. © 2011 Wiley Periodicals, Inc.