Voltage-gated Na v channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na v 1.5 is the predominant Na v channel, and Na v 1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na v 1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na v channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na v 1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na v 1.5 expression, abnormal Na v 1.5 membrane targeting, and reduced Na + channel current density. We define the structural requirements on ankyrin-G for Na v 1.5 interactions and demonstrate that loss of Na v 1.5 targeting is caused by the loss of direct Na v 1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Na v channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na v channel organization in multiple excitable tissues.