Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease associated with significant morbidity and mortality in humans. However, the molecular etiology underlying TOF in most patients remains largely unknown. In the present study, sequence analysis of the GATA6 gene was performed from fresh-frozen cardiac tissues and matched blood samples of 52 unrelated patients who underwent surgical repair of TOF. The cardiac tissues and matched blood specimens from 46 patients who underwent cardiac valve replacement due to rheumatic heart disease and blood samples from 200 healthy individuals as controls were genotyped. The functional characteristics of the mutations were assessed using a luciferase reporter assay system. Based on the results, two novel heterozygous GATA6 mutations, p.G367X and p.G394C, were identified in the cardiac tissues of 2 TOF patients, respectively. No mutations were found in the cardiac tissues from 46 patients with rheumatic heart disease and in the blood samples from the 298 participants. Functional analysis demonstrated that the GATA6 mutants were consistently associated with significantly reduced transcriptional activation compared with their wild-type counterpart. This is the first report on the link of somatic GATA6 mutation to TOF, providing novel insight into the molecular mechanism involved in TOF.