Azidothymidine(AZT)is an antiretroviral drug that affects cell proliferation, apoptosis, and the NF-κB pathway. As multiple myeloma (MM)presents with constitutive activation of NF-κB, we analyzed the effect of AZT on human MM cell lines. We evaluated the cytotoxic effect of AZT in human MM cell lines sensitive (8226/S)orresistant to doxorubicin (8226/DX5) and human T cell lymphoblast-like cells, uterine sarcoma cells, and HUVEC using MTT assay. Cytotoxicitywas also evaluated in vivo in nude mice xenografted with an8226/S tumor. The effect of AZT on the expression of genes involved in cell proliferation, apoptosis, angiogenesis, and the NF-κB pathway was analyzed in the xenografts usingreal-time polymerase chain reaction.AZT was effective against both 8226/S and 8226/DX5 cells in a dose and time-dependent manner(p = 0.02)in vitro and promoted cell cycle arrest in S phase in these cells. The tumor volume was lower in mice treated with AZT compared tountreated mice (p = 0.0003). AZT down-regulated the pro-proliferative genes encodingAKT1, MYC, STAT1, MAPK8, MAPK9, CCL-3, Bcl-3, and cyclin D2;pro-angiogenenic genes encodingVEGF and IL8; and genes involved in cell adhesion (ICAM1 and FN1) and the NF-κBpathway. AZT up-regulated the expression of tumor suppressor gene FOXP1and the pro-apoptotic genes encoding BID, Bcl-10, and caspase-8. Thus, we demonstrated the cytotoxic effect of AZT in human MM cell lines for the first time. Our data may provide the rationale for future clinical trials of AZT for treating MM.