The high mobility group A2 (HMGA2) protein belongs to the architectural transcription factor HMGA family, playing a role in chromosomal organization and transcriptional regulation. We and others have previously reported that ectopic HMGA2 expression is associated with neoplastic transformation and anchorage-independent cell proliferation. Here, we reported a correlation between increased HMGA2 expression and enhanced chemosensitivity towards topoisomerase II inhibitor, doxorubicin, in breast cancer cells. Using cells exhibiting differential HMGA2 expression and small interfering RNA technique, we showed that HMGA2 expression modulates cellular response to the genotoxicity of DNA double-strand breaks. Notably, HMGA2 enhances doxorubicin-elicited cell cycle delay in sub-G1 and G2-M and augments cell cycle dysregulation on cotreatment of doxorubicin and caffeine. We further reported that HMGA2 induces a persistent Ser139 phosphorylation of histone 2A variant X, analogous to the activation by doxorubicin-mediated genotoxic stress. Moreover, this HMGA2-dependent enhancement of cytotoxicity is further extended to other double-strand breaks elicited by cisplatin and X-ray irradiation and is not restricted to one cell type. Together, we postulated that the enhanced cytotoxicity by double-strand breaks in HMGA2-expressing cells is mediated, at least in part, through the signaling pathway of which the physiologic function is to maintain genome integrity. These findings should contribute to a greater understanding of the role of HMGA2 in promoting tumorigenesis and conveying (chemo)sensitivity towards doxorubicin and other related double-strand breaks.