Abstract. Arsenic hexoxide (As 4 O 6 ) has been used in Korean folk remedy for the treatment of cancer since the late 1980s. Evidence suggests that the anticancer effects of As 4 O 6 are different from those of As 2 O 3 . Tumor necrosis factor-α (TNF-α) is generally increased in advanced cancer and is closely related to cancer progression, although it has cancer-killing effects. The reason is that TNF-α activates nuclear factor-κB (NF-κB) that is involved in cell proliferation, invasion, drug resistance and metastasis. In the present study, we investigated the effects of As 4 O 6 on NF-κB activity, NF-κB-mediated cellular responses, and NF-κB-regulated gene expressions involved in metastasis at the concentrations of As 4 O 6 where no cytotoxicity was observed. As 4 O 6 suppressed NF-κB activation in both TNF-α-treated and control cells, and also suppressed IκB phosphorylation in a time-dependent manner, suggesting the suppression of NF-κB results, in part, from the inhibition of IκB degradation. We also confirmed the anti-NF-κB activity of As 4 O 6 with synergism with TNF-α by augmenting caspase-8 activation. As 4 O 6 also suppressed NF-κB activation induced by TNF-α, and some of the downstream NF-κB-regulated proteins involved in cancer proliferation, anti-apoptosis and metastasis. In conclusion, the present study demonstrated that As 4 O 6 has anticancer properties by inhibiting NF-κB activation and NF-κB-regulated proteins at least in part through the inhibition of IκB phosphorylation, especially in the conditions of advanced cancer where TNF-α is highly secreted.