High mobility group A1 (HMGA1) protein is a non-histone chromatin binding protein, which acts as an architectural transcriptional factor and participates in transcriptional regulation, transformation and various cellular processes. Emerging evidences suggest that hmga1 gene is highly expressed in all aggressive human cancers studied to date and is considered an attractive target for therapeutic intervention. In this study, we investigated the interaction of triplex forming oligonucleotide (TFO), targeted to its cognate site at position, -1917 to -1940 located within a regulatory element of hmga1 promoter and its consequent effect on the expression of HMGA1 in human cervical cancer cells. Two sequential melting transitions as revealed by UV-Vis, Circular Dichroic spectroscopy suggested stable DNA triplex formation which was further authenticated by gel retardation assay using γ-32P [ATP]. Thermodynamics of the DNA triplex was studied by isothermal titration and differential scanning calorimetry that demonstrated the binding of TFO to the targeted gene sequence to be exothermic in nature and favored by both negative enthalpy (∆H, -91.16±0.3 kcalM-1) and positive entropy changes (T∆S, 81.05±0.1 kcalM-1). Treatment of HeLa cells with hmga1-TFO as therapeutics modality significantly reduced the expression of HMGA1, both at transcriptional as well as translational level (~50%) associated with inhibition of cell proliferation as compared to control TFO. Our findings suggest that targeting hmga1 gene is a promising strategy for the development of DNA triplex-based novel anti-cancer therapeutics.