The cardiac sodium channel Nav1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Nav1.5 C-terminus. Pull-down experiments were performed using Nav1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx5cv mice to study the role of this protein complex in Nav1.5 function. Western blot experiments revealed a 50% decrease in the Nav1.5 protein levels in mdx5cv hearts, whereas Nav1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx5cv cardiomyocytes. Finally, ECG measurements of the mdx5cv mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Nav1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.