Atrial fibrillation (AF) represents the most common form of sustained cardiac arrhythmia and accounts for substantial morbidity and mortality. Mutations in the cardiac sodium channel α, β1, β2 and β3 subunit genes (SCN5A, SCN1B, SCN2B and SCN3B) have been associated with AF, which suggests that mutations in the sodium channel β4 subunit gene, SCN4B, are also involved in the pathogenesis of AF. To examine this hypothesis, the coding exons and exon-intron boundaries of SCN4B were sequenced in 170 unrelated index patients with familial AF. The available relatives of the probands carrying the identified mutations and 200 unrelated ethnically matched healthy individuals used as the controls were subsequently genotyped. The pathogenic potential of a SCN4B sequence variation was predicted using MutationTaster. As a result, 2 novel heterozygous SCN4B mutations, p.V162G and p.I166L, were identified in 2 unrelated families with AF transmitted in an autosomal dominant pattern, respectively. In each family the mutation co-segregated with AF and was absent in the 400 control chromosomes. The mutations altered the amino acids evolutionarily highly conserved across species and were both predicted to be disease-causing. To the best of our knowledge, this is the first study to demonstrate an association of SCN4B mutations with AF, suggesting SCN4B as a novel AF susceptibility gene.