Our study aims to investigate the impact of the Maturity-onset diabetes of the young 3 disease-linked rSNP rs35126805 located in the HNF-1α gene promotor on the binding of the transcription factor HNF-4α and consequently on the regulation of HNF-1α gene expression. Our focus is to calculate the change in the binding affinity of the transcription factor HNF-4α to the DNA, caused by the regulatory single nucleotide polymorphism (rSNP) through molecular dynamics simulations and thermodynamic analysis of acquired results. Both root-mean-square difference (RMSD) and the relative binding free energy ΔΔGbind reveal that the HNF-4α binds slightly more strongly to the DNA containing the mutation (rSNP) making the complex more stable/rigid, and thereby influencing the expression of the HNF-1α gene. The resulting disruption of the HNF-4α/HNF-1α pathway is also linked to hepatocellular carcinoma metastasis and enhanced apoptosis in pancreatic cancer cells. To the best of our knowledge, this represents the first study where thermodynamic analysis of the results obtained from molecular dynamics simulations is performed to uncover the influence of rSNP on the protein binding to DNA. Therefore, our approach can be generally applied for studying the impact of regulatory single nucleotide polymorphisms on the binding of transcription factors to the DNA.