Ventricular septal defect (VSD) is the most common type of congenital heart disease. HAND1 gene plays a crucial role in the development of the heart, but the role of the variants in the HAND1 gene promoter region in patients with VSD has not been explored yet. From 588 participants (300 with isolated and sporadic VSD and 288 healthy controls), DNA was extracted from blood samples. Variants at the HAND1 gene promoter region were analyzed through Sanger sequencing. Subsequently, cell functional validation was conducted through cell experiments, including dual-luciferase reporter gene analysis, electrophoretic mobility shift analysis, and bioinformatics analysis was also conducted. The promoter region of HAND1 gene had a total of 9 identified variant sites. Among them, 4 variants were exclusively found in VSD patients, and 1 variant (g.3631A>C) was newly discovered. Cell functional experiments indicated that all four variants decreased the transcriptional activity of HAND1 gene promoter with three of them reached statistical significance (p < 0.05). Subsequent analysis using JASPAR (a transcription factor binding profile database) suggests that these variants may alter the binding sites of transcription factors, potentially contributing to the formation of VSD. Our study for the first time identified variants in the promoter region of HAND1 gene in Chinese patients with isolated and sporadic VSD. These variants significantly decreased the expression of HAND1 gene, impacting transcription factor binding sites, and thereby demonstrating pathogenicity. This study offers new insights into the role of HAND1 gene promoter region, contributing to a better understanding of the genetic basis of VSD formation.