Genome-wide association studies (GWASs) have successfully identified numerous non-coding genetic variants for type 2 diabetes (T2D), but the functional roles underlying these non-coding variants remain largely unknown. The effects of T2D GWAS lead SNPs on transcriptional factors binding motifs were firstly analyzed via JASPAR, followed by functional validations including dual-luciferase reporter assays, biotin-based DNA pull-down assays, real-time quantitative PCR, and western blotting. The results showed that GWAS SNP rs4430796 conferred T allele specific transcriptional enhancer activity via a PAX6 binding element, and upregulated the expression of HNF1B. GWAS SNP rs4607103 showed a bidirectional modulation of ADAMTS9-AS2 and ADAMTS9 by TCF7L2 in a T allele-specific manner. GWAS SNP rs849135 conferred C allele-specific bidirectional transcriptional enhancer activity via a CREB1 binding element. Our findings have uncovered the functional mechanisms of three T2D GWAS SNPs via affecting the binding of transcription factors, providing new insights into the genetics and molecular pathogenesis of T2D. (C) 2020 Elsevier Inc. All rights reserved.