Tetraarsenic hexoxide (As4O6) has been used in Korean traditional medicine for the treatment of cancer since the late 1980's, and arsenic trioxide (As2O3) is currently used as a chemotherapeutic agent. Previous studies suggest that the As4O6-induced cell death pathway is different from that of As2O3 and its mechanism of anticancer activity remains unclear. Nuclear factor (NF)-κB is a well-known transcription factor involved in cell proliferation, invasion and metastasis. Hence, in the present study, we investigated the effects of As4O6 on NF-κB activity and NF-κB-regulated gene expression in vitro and in vivo. The cytotoxicity assay revealed that As4O6 inhibited the growth of SW620 cells in a dose-dependent manner, and the half maximal inhibitory concentration (IC50) was ~1 µM after a 48 h treatment. As4O6 suppressed NF-κB activation and suppressed inhibitory κBα (IκBα) phosphorylation stimulated by tumor necrosis factor (TNF). As4O6 also suppressed downstream NF-κB-regulated proteins involved in cancer anti-apoptosis, proliferation, invasion and metastasis. In addition, As4O6 marginally suppressed tumor growth and the anti-NF-κB activity was confirmed using an in vivo xenograft mouse model in which animals were injected with SW620 cells. The present study provides evidence that As4O6 has anticancer properties through suppression of NF-κB activity and NF-κB-mediated cellular responses.