The cardiac sodium channel Na_v1.5 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 Na_v1.5 C-terminus. Pull-down experiments were performed using Na_v1.5 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 mdx^5cv mice to study the role of this protein complex in Na_v1.5 function. Western blot experiments revealed a 50% decrease in the Na_v1.5 protein levels in mdx^5cv hearts, whereas Na_v1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx^5cv cardiomyocytes. Finally, ECG measurements of the mdx^5cv 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 Na_v1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.