Stromal cell-derived factor-1 (SDF-1) plays a role in the homing of myeloma cells to bone marrow. In addition, SDF-1 modestly enhances the proliferation of myeloma cells and inhibits Dexmethasone (Dex)-induced apoptosis of the cells. Dex is currently used to treat multiple myeloma, based on its apoptic effects. In this study, we investigated the regulatory effects of Dex on SDF-1 production in bone marrow stromal cells (BMSCs) and on CXCR4 expression in myeloma cells. As previously reported, it was evident that primary myeloma cells (CD138+ cells obtained from patients with multiple myeloma) and Dex-resistant myeloma cell line RPMI8226 expressed CXCR4 and responded to SDF-1, resulting in chemotaxis. SDF-1 modestly stimulated the proliferation of primary myeloma cells and RPMI8226 cells and protected the cells from Dex-induced apoptosis. Human umbilical vein endothelial cells transduced with the SDF-1 gene using adenoviral vectors better supported the formation of cobblestone areas of primary myeloma cells and RPMI8226 cells in co-culture, similar to hematopoietic progenitor cells; this was blocked by pretreating the myeloma cells with pertussis toxin, indicating that SDF-1 plays a critical role not only in migration of the cells underneath the SDF-1-producing stromal cells but also in proliferation of the cells in contact. Dex up-regulated CXCR4 expression in RPMI8226 cells; however, its regulatory effects on CXCR4 in primary myeloma cells differed among patients. RT-PCR and Northern blot analyses revealed that Dex down-regulated SDF-1 mRNA expression in both primary BMSCs and murine stromal MS-5 cells in a dose-dependent manner. Western blot analysis and ELISA assay confirmed that Dex inhibited SDF-1 production in BMSCs. Furthermore, Dex inhibited cobblestone area formation of RPMI8226 cells in co-culture with MS-5. Interestingly, Dex up-regulated CXCR4 mRNA expression and cytoplasmic CXCR4 in BMSCs. These results indicate that Dexamethasone induces the down-regulation of SDF-1 production in BMSCs, which might mediate, at least in part, its anti-myeloma effects in vivo.