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.