Y-box binding protein 1 (YB-1) is a member of the large protein family with an evolutionarily ancient coldshock domain. It is involved in the regulation of some global traits of malignancy and functions both in the cell nucleus and cytoplasm. We investigated the content of YB-1 mRNA and proportion of cells with YB-1 nuclear localization in breast cancer (BC) tumors taken at mastectomy. We showed that an increased level of YB-1 mRNA in samples correlated with tumor aggressiveness and increased expression of multidrug resistance (MDR) genes. Over-expression of several MDR genes was found in the samples with high YB-1 mRNA content and with YB-1 cytoplasmic localization. This suggests that YB-1 regulates expression of some MDR genes at the level of cytoplasm. The nuclear YB-1 localization also correlated with BC aggressiveness; however, these interconnections were less prominent in comparison with those between a high YB-1 mRNA content and the mentioned tumor characteristics. Our results show that neo-adjuvant chemotherapy induces translocation of YB-1 from the cytoplasm to the cell nucleus in mammary tumors. In the experiments with cell cultures we showed that chemotherapeutic drugs stimulated YB-1 nuclear translocation, as well as MDR genes expression and cell multidrug resistance. We suppose that YB-1 nuclear translocation determines adaptation of tumor cell populations to the environmental impact, while a high YB-1 mRNA content is an intrinsic feature that contributes to the control of the global traits of malignancy. A combination YB-1 mRNA content and its intracellular localization may serve as a test for BC prognosis.