The SCN5A gene encodes cardiac sodium channel Na_v1.5 and causes lethal ventricular arrhythmias/sudden death and atrial fibrillation (AF) when mutated. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and involved in the pathogenesis of many diseases. However, little is known about the regulation of SCN5A by miRNAs. Here we reveal a novel post-transcriptional regulatory mechanism for expression and function of SCN5A/Na_v1.5 via miR-192-5p. Bioinformatic analysis revealed that the 3′-UTR of human and rhesus SCN5A, but not elephant, pig, rabbit, mouse, and rat SCN5A, contained a target binding site for miR-192-5p and dual luciferase reporter assays showed that the site was critical for down-regulation of human SCN5A. With Western blot assays and electrophysiological studies, we demonstrated that miR-192-5p significantly reduced expression of SCN5A and Na_v1.5 as well as peak sodium current density I_Na generated by Na_v1.5. Notably, in situ hybridization, immunohistochemistry and real-time qPCR analyses showed that miR-192-5p was up-regulated in tissue samples from AF patients, which was associated with down-regulation of SCN5A/Na_v1.5. These results demonstrate an important post-transcriptional role of miR-192-5p in post-transcriptional regulation of Na_v1.5, reveal a novel role of miR-192-5p in cardiac physiology and disease, and provide a new target for novel miRNA-based antiarrhythmic therapy for diseases with reduced I_Na.