ABSTRACT Talin (tln) binds and activates integrins to couple extracellular matrix‐bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1 fl02k mutants. The ethylnitrosourea‐induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1 , but neither tln2 nor ‐2a , was dominantly expressed in the heart and vessels. Unlike tln1 and ‐2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z‐disks and endothelial/endocardial cell integrity, partly through regulating F‐actin networks in zebrafish. The similar expression profiles of tln1 and integrin β1b ( itgb1b ) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z‐disks and vessel lumens. Taken together, the results of this work suggest that Tln1‐mediated Itgβ 1b plays a crucial role in maintaining cardiac sarcomeric Z‐disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.—Wu, Q., Zhang, J., Koh, W., Yu, Q., Zhu, X., Amsterdam, A., Davis, G. E., Arnaout, M. A., Xiong, J.‐W. Talin1 is required for cardiac Z‐disk stabilization and endothelial integrity in zebrafish. FASEB J. 29, 4989–5005 (2015). www.fasebj.org