Atrial septal defect (ASD) is a common type of congenital heart disease, which is defined as any communication through atrial septum. Several studies have revealed that genetic factors may influence the susceptibility of ASD. Recent studies have shown that reticulon 4 (RTN4) gene might be involved in some processes relevant to heart development, such as regulation of cell migration and vascular remodeling. This study aimed to evaluate RTN4 gene polymorphisms of CAA and TATC insertion/deletion in relation to the risk of ASD in Chinese Han population. A total of 175 ASD patients and 308 unrelated healthy controls were successfully investigated. The polymorphisms of patients were determined by polymerase chain reaction-polyacrylamide gel electrophoresis. There was no significant difference in the allele frequencies of CAA and TATC insertion/deletion in RNT4 gene between ASD patients and controls. The same results were seen in their genotypes. The present study suggests that CAA and TATC insertion/deletion polymorphisms of RNT4 gene may not be a useful marker to predict the susceptibility of ASD in Chinese Han population.