Arsenic trioxide (As(2)O(3)) has shown remarkable efficacy for the treatment of multiple myeloma (MM). Histone deacetylases (HDAC) play an important role in the control of gene expression, and their dysregulation has been linked to myeloma. Especially, HDAC6, a unique cytoplasmic member of class II, which mainly functions as α-tubulin deacetylase and Hsp90 deacetylase, has become a target for drug development to treat cancer due to its major contribution in oncogenic cell transformation. However, the mechanisms of action for As(2)O(3) have not yet been defined. In this study, we investigated the effect of As(2)O(3) on proliferation and apoptosis in human myeloma cell line and primary myeloma cells, and then we studied that As(2)O(3) exerts antimyeloma effects by inhibiting activity in the α-tubulin and Hsp90 through western blot analysis and immunoprecipitation. We found that As(2)O(3) acts directly on MM cells at relatively low concentrations of 0.5~2.5 µM, which effects survival and apoptosis of MM cells. However, As(2)O(3) inhibited HDAC activity at the relatively high concentration and dose-dependent manner (great than 4 µM). Subsequently, we found that As(2)O(3) treatment in a dose- and time-dependent fashion markedly increased the level of acetylated α-tubulin and acetylated Hsp90, and inhibited the chaperone association with IKKα activities and increased degradation of IKKα. Importantly, the loss of IKKα-associated Hsp90 occurred prior to any detectable loss in the levels of IKKα, indicating a novel pathway by which As(2)O(3) down-regulates HDAC6 to destabilize IKKα protein via Hsp90 chaperone function. Furthermore, we observed the effect of As(2)O(3) on TNF-α-induced NF-κB signaling pathway was to significantly reduced phosphorylation of Ser-536 on NF-κB p65. Therefore, our studies provide an important insight into the molecular mechanism of anti-myeloma activity of As(2)O(3) in HDAC6-Hsp90-IKKα-NFκB signaling axis and the rationale for As(2)O(3) can be extended readily using all the HDAC associated diseases.