DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.