Establishing correct left-right asymmetry during embryonic development is crucial for proper asymmetric positioning of the organs. Congenital heart defects such as dextrocardia, transposition of the arteries, and inflow or outflow tract malformations, comprise some of the most common birth defects and may be attributed to incorrect establishment of body laterality. Here we identify 3 new patients with dextrocardia who have mutations in CFAP53, a coiled-coil domain containing protein. To elucidate the mechanism by which CFAP53 regulates embryonic asymmetry we used genome editing to generate cfap53 zebrafish mutants. Zebrafish cfap53 mutants have specific defects in organ laterality, and randomisation of asymmetric gene expression. We show that cfap53 is required for cilia rotation specifically in Kupffer's vesicle, the zebrafish laterality organ, providing a mechanism by which patients with CFAP53 mutations develop dextrocardia and heterotaxy, and confirming previous evidence that left-right asymmetry in humans is regulated through cilia-driven fluid flow in a laterality organ. This article is protected by copyright. All rights reserved.