2-Methoxyestradiol (2ME2) an estrogen derivative, induces growth arrest and apoptosis in leukemic cells and is also antiangiogenic. In this study, we demonstrate that 2ME2 inhibits growth and induces apoptosis in multiple myeloma (MM) cell lines and patient cells. Significantly, 2ME2 also inhibits growth and induces apoptosis in MM cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40 and Dox-6), and dexamethasone (MM.1R). In contrast to its effects on MM cells, 2ME2 does not reduce the survival of normal peripheral blood lymphocytes. Moreover, 2ME2 enhances Dex-induced apoptosis, and its effect is not blocked by interleukin-6 (IL-6). We next examined the effect of 2ME2 on MM cells in the bone marrow (BM) milieu. 2ME2 decreases survival of BM stromal cells (BMSCs), as well as secretion of vascular endothelial growth factor (VEGF), and IL-6 triggered by the adhesion of MM cells to BMSCs. We show that apoptosis induced by 2ME2 is mediated by the release of mitochondrial cytochrome-c (cyto-c) and Smac, followed by the activation of caspases-8, -9, and -3. Finally, 2ME2 inhibits MM cell growth, prolongs survival, and decreases angiogenesis in a murine model. These studies, therefore, demonstrate that 2ME2 mediates anti-MM activity directly on MM cells and in the BM microenvironment. They provide a framework for the use of 2ME2, either alone or in combination with Dex, to overcome drug resistance and to improve outcome in MM.