Technical Abstract: Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR) process. Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ataxia telangiectasia mutated (ATM) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine and sodium selenite via reactive oxygen species (ROS), and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G2/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidants N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses the HMLH1 dependent DNA damage responses to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 (a heterodimer protein complex critical for functional MMR) that is dependent on ATM and ROS. Taken together, our results describe a novel role for selenium in mitigating tumorigenesis by promoting this critical dimer formation followed by DNA repair. Lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.