Integrins and proteins that associate with integrins are implicated in normal cardiac muscle function and development. Unc-112 is a cytoplasmic adaptor protein required for the proper establishment of integrin junctions in Caenorhabditis elegans muscle. A vertebrate homolog of unc-112, kindlin-2, is an integrin-binding protein that is expressed in cardiac muscle, but its function is unknown. We sought to understand the role of kindlin-2 in the development and function of the mouse and zebrafish heart. In the mouse, we found that kindlin-2 is highly expressed in the heart and is enriched at intercalated discs and costameres. Targeted disruption of the murine kindlin-2 gene resulted in embryonic lethality before cardiogenesis. To better assess the role of kindlin-2 in cardiac muscle development, we used morpholinos to knockdown the kindlin-2 homolog in zebrafish (z-kindlin-2), which resulted in severe abnormalities of heart development. Morphant hearts were hypoplastic and dysmorphic and exhibited significantly reduced ventricular contractility. Ultrastructural analysis of these hearts revealed disrupted intercalated disc formation and a failure in the attachment of myofibrils to membrane complexes. We conclude that kindlin-2 is an essential component of the intercalated disc, is necessary for cytoskeletal organization at sites of membrane attachment, and is required for vertebrate myocardial formation and function. These findings provide the first characterization of the in vivo functions of this novel and critical regulator of cardiogenesis.