The high mobility group A1 (HMGA1) are non-histone chromosomal proteins consisting of HMGA1a and HMGA1b which act as architectural transcription factors. Elevated levels of HMGA1 are reported in a number of human cancers and suggested as tumor markers. Due to their role in neoplastic transformation and tumor progression, we considered HMGA1 as a potential target for downregulation at the transcriptional level. The present paper deals with the binding of a widely used chemotherapeutic agent, adriamycin (ADM), to hmga1gene promoter (−304 to −284), 21RY, and its effect on the expression of hmga1 at the mRNA and protein levels and further its cytotoxic efficacy in A431 cells. A strong complex (21RY–ADM) formation caused hypochromic and bathochromic changes in UV-absorption, considerable spectral changes in circular dichroism of adriamycin and DNA and significant quenching of fluorescence emission of ADM. Thermodynamics of 21RY–ADM interaction was studied by isothermal titration and differential scanning calorimetric techniques that revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. Further, even low concentrations (10.3 nM) of ADM showed cytotoxicity on human squamous carcinoma cells (A431) and caused downregulation (by ∼70%) of hmga1 at mRNA and protein levels. Present findings clearly support the inhibitory effect of ADM on hmga1 which quite probably is the consequence of its binding to the targeted region of hmga1. Therefore, it appears that hmga1 is a novel potential chemotherapeutic target in treating carcinomas of epithelial origin like prostate, breast, thyroid etc.