NaV1.5 is a cardiac voltage-gated Na+ channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of NaV1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5′-CAAAACA-3′ (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5′-CAAAACAAAACA-3′. This finding led us to hypothesize that Foxo1 regulates NaV1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates NaV1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H2O2)-mediated NaV1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of NaV1.5 expression and Na+ current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of NaV1.5 expression. H2O2 significantly reduced NaV1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates NaV1.5 expression in cardiomyocytes and reactive oxygen species suppress NaV1.5 expression through Foxo1.