Runt domain transcription factor 3 (RUNX3) is a tumor suppressor that is silenced in cancer via hypermethylation of its promoter. This study investigated the mechanisms involved in reactive oxygen species (ROS)-induced silencing of RUNX3 in terms of epigenetic alteration since the effects of oxidative stress in tumor suppressor gene transcription are largely unknown. RUNX3 mRNA and protein expressions were down-regulated in response to hydrogen peroxide (H(2)O(2)) in the human colorectal cancer cell line SNU-407. This down-regulation was abolished with pretreatment of the ROS scavenger, N-acetylcysteine (NAC). Moreover, methylation-specific PCR data revealed that H(2)O(2) treatment increased RUNX3 promoter methylation; however, NAC and the cytosine methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC), decreased it, suggesting that an epigenetic regulatory mechanism by ROS-induced methylation may be involved in RUNX3 silencing. H(2)O(2) treatment resulted in DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) up-regulation with increased expression and activity, increased binding of DNMT1 to HADC1, and increased DNMT1 binding to the RUNX3 promoter. In addition, 5-Aza-dC treatment prevented the decrease in RUNX3 mRNA and protein levels by H(2)O(2) treatment. Additionally, H(2)O(2) treatment inhibited the nuclear localization and expression of RUNX3, which was abolished by NAC treatment. Furthermore, the down-regulation of RUNX3 expression by H(2)O(2) also influenced cell proliferation. Taken together, the data suggested that ROS silenced the tumor suppressor, RUNX3, by epigenetic regulation and may therefore be associated with the progression of colorectal cancer.