We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 μmol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 μmol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 μmol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death. [Cancer Res 2008;68(7):2358–65]