Y-box-binding protein-1 (YB-1) is a multifunctional protein involved in the regulation of transcription, translation, and mRNA splicing. In recent years, several laboratories have demonstrated that YB-1 is directly involved in the cellular response to genotoxic stress. Importantly, YB-1 is increased in tumor cell lines resistant to cisplatin, and the level of nuclear expression of YB-1 is predictive of drug resistance and patient outcome in breast tumors, ovarian cancers, and synovial sarcomas. YB-1 binds to several DNA repair enzymes in vitro including human endonuclease III (hNTH1). Human NTH1 is a bifunctional DNA glycosylase/apurinic/apyrimidinic lyase involved in base excision repair. In this study, we show that YB-1 binds specifically to the auto-inhibitory domain of hNTH1, providing a mechanism by which YB-1 stimulates hNTH1 activity. Indeed, YB-1 strongly stimulates in vitro the activity of hNTH1 toward DNA duplex probes containing oxidized bases, lesions prone to be present in cisplatin treated cells. We also observed an increase in YB-1/hNTH1 complex formation in the mammary adenocarcinoma MCF7 cell line treated with UV light and cisplatin. Such an increase was not observed with mitomycin C or the topoisomerase I inhibitor camptothecin. Accordingly, antisense RNAs against either YB-1 or hNTH1 increased cellular sensitivity to UV and cisplatin but not to mitomycin C. An antisense RNA against YB-1 increased camptothecin sensitivity. In contrast, an antisense against hNTH1 did not. Finally, siRNA against hNTH1 re-established cytotoxicity in otherwise cisplatin-resistant YB-1 overexpressing MCF7 cells. These data indicate that hNTH1 is a relevant target to potentiate cisplatin cytotoxicity in YB-1 overexpressing tumor cells.