YB-1 is a protein involved in DNA repair, transcription, splicing, translation, and confers cisplatin resistance in several cancers. However, it is unknown which YB-1 activity is required for this resistance. To identify the mechanism(s) by which nuclear YB-1 confers cisplatin resistance, we generated several YB-1 mutants and tested their impact on resistance in the mammary tumor cell lines MCF7 and MDA-MB-231. Transfection of wild type YB-1 bestowed cisplatin resistance in such cells but a mutant YB-1 with a point mutation at position 175 (YB-1(E175A)) did not. A truncated YB-1(1-205) increased cisplatin resistance above the levels conferred by wild type YB-1. The truncated YB-1(1-205) has intact nuclease activities but could not separate a DNA duplex containing a Y-box sequence (activities associated with DNA repair). Moreover, this truncated YB-1(1-205) did not alter splicing of the adenovirus E1A pre-mRNA minigene as it had low binding affinity for several splicing factors. In contrast, the mutant YB-1(E175A) protein behaved like wild type YB-1 regarding all these activities but yet did not confer cisplatin resistance. Finally, transfection of mutant YB-1(E175A) had low impact on overall transcription. The wild type and truncated YB-1(1-205) induced important but different alterations in gene expression as revealed by microarray analyses. Our results indicate that the splicing and the nuclease activities associated with YB-1 have minor impact on cisplatin resistance. In contrast, the global expression profiles displayed by both wild type and truncated YB-1(1-205) revealed several chemoresistance signatures which differed depending on the genetic status of the breast cancer cell line used.