Trabectedin (Ecteinascidin-743 or ET-743) is a novel antitumour agent of marine origin with potent antitumour activity both in vitro and in vivo. It interacts with the minor groove of DNA, interfering with transcriptional activity and DNA repair pathways. Here, we report a novel mechanism by which trabectedin exerts its cytotoxic effects on carcinoma cells. It is based on its ability to impair the function of the High-Mobility Group A (HMGA) proteins. These proteins have a key role in cell transformation, and their overexpression is a common feature of human malignant neoplasias, representing a poor prognostic index often correlated to anti-cancer drug resistance. They bind the minor groove of DNA, alter chromatin structure and, thus, regulate the transcription of several genes by enhancing or suppressing the activity of transcription factors. We first report that trabectedin has a higher cytotoxic effect on thyroid and colon carcinoma cells expressing abundant levels of HMGAs in comparison with cells not expressing them. Then, we have shown that trabectedin treatment displaces HMGA proteins from the HMGA-responsive promoters, including ATM promoter, impairing their transcriptional activity. Finally, we report a synergism between Ionising Radiations and trabectedin treatment restricted to the HMGA-overexpressing cancer cells. This result might have important clinical implications since it would suggest the use of trabectedin for the treatment of neoplasias expressing abundant HMGA levels that are frequently associated to chemoresistance and poor prognosis.