Resistance to chemotherapy is responsible for failure of current treatment regimens in cancer patients. We have previously reported that nuclear localization of the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1 which plays a major role in the development of a multidrug resistant tumor phenotype. Recent research has indicated that adenoviruses as oncolytic agents could be used as an alternative therapeutic approach in cancer patient. However, successful therapy crucially requires understanding how viral replication is brought about. Recently we were able to demonstrate that nuclear accumulation of the human transcription factor YB-1 in adenovirus infected cells is a function of the E1-region and we have shown that YB-1 facilitates adenovirus replication. Here we report that in multidrug resistant cancer cells, where YB-1 is located in the nucleus, adenovirus vectors such as Ad312 and dl520 efficiently replicate and induce strong cell lysis in vitro and in vivo. Thus replication defective adenoviruses are a previously unrecognized vector system for a selective elimination of multidrug resistant cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors in the treatment of multidrug resistant malignant diseases in the clinical setting.