The nuclear erythroid factor 2-related factor 2 (Nrf2) is widely known as an important redox factor to protect cells from oxidative stress. However, recently it is known that Nrf2 in cancer cells provides advantages for cell viability from the exposure to anticancer drugs. Mitomycin C (MMC) is an anticancer drug for treatment of several types of cancers including colon and breast cancers. It is potent DNA crosslinker. MMC is able to induce ROS as part of its mechanism of action, which exerts detrimental effects to cancer cells. In this study, we investigated the role of Nrf2 in resistance to MMC in human colon cancer cells. Stable Nrf2 shRNA-expressing RKO showed higher sensitivity to MMC exposure than Nrf2 wild type cells. Furthermore, significantly increased apoptosis was associated with high level of oxidative stress and expression of DNA damage sensitive protein, phosphorylated H2AX (gamma-H2AX) in Nrf2 deficient human colon RKO cells. In conclusion, we demonstrated for the first time that Nrf2 enhanced MMC-resistance via inhibition of oxidative stress and DNA damage in human colon cancer cells. Therefore, we suggested that Nrf2 might be potential therapeutic target in the MMC treatment of colon cancer.