Objectives A number of risk variants have been identified from Genome-wide association studies (GWAS) of Congenital Heart Disease (CHD). However, none of them has been functionally confirmed as yet. We sought to identify the gene and mechanism responsible for the GWAS signal (OR=1.46; p=2.61×10–10) which we previously identified at chromosome 4p16 for atrial septal defect (ASD). Methods and results Exploration of the linkage disequilibrium pattern between SNPs in the region indicated association was restricted to the interval spanning the long noncoding RNA STX18-AS1. Since no homolog of STX18-AS1 is found beyond primates, all experiments were conducted in human tissues and cell lines. With RNA from 108 Right Atrial Appendages and corresponding blood DNA, we confirmed the risk SNPs (rs870142 and rs16835979) were eQTLs for STX18-AS1 in human cardiac tissues. In RNA expression analyses using qPCR on embryonic heart samples, the transcription of STX18-AS1 was detected to be the highest at CS14-CS18, the critical time for atrial septation during human heart development. With in situ hybridization on whole embryonic hearts of CS17-CS19, the expression of STX18-AS1 was also determined in the myocardium of Atrial Septum. We next identified STX18-AS1 as a regulator of the key cardiac transcriptional factor NKX2-5 using CRISPR/Cas9 knockdown of STX18-AS1 in HepG2 cells. Mutations in NKX2-5 cause septal defects in humans. Reduced STX18-AS1 transcription inhibited the expression of NKX2-5 accompanied by decreased H3K4me3 at its promoter region. We demonstrated the interaction between STX18-AS1 RNA and WDR5 protein, which supported the epigenetic regulation effects of STX18-AS1. Using in vitro cardiomyocyte differentiation from human embryonic stem cells, we demonstrated that the knockdown of STX18-AS1 depleted the potential of human embryonic stem cells in differentiating into beating cardiomyocytes without changes in cell viability and pluripotency. Conclusion STX18-AS1 is the first long noncoding RNA influencing CHD risk identified from GWAS. The mechanism involves downregulation of the NKX2-5 gene through epigenetic mechanisms. Conflict of interest No