Thioredoxin reductase 1 (Trr1) is an antioxidant and redox regulator that functions in governing the cellular redox state and survival against oxidative insults in mammals. However, this selenoprotein is also overexpressed in various forms of malignant cancers, leading to the hypothesis that Trr1 may be a potential target for cancer therapy. A quinone anti-cancer drug, mitomycin C (MMC), has been clinically used in the treatment of several types of tumors, including those of the colon. MMC exerts its activity via ROS induction and further results in DNA cross-linkage. To evaluate the significant role of Trr1 in MMC resistance in human colon cancer (RKO) cells, specific reduction in the expression of Trr1 was achieved using short-hairpin RNA (shRNA)-based interference. Our results showed that stable Trr1 shRNA knockdown manifested higher cellular susceptibility to MMC in comparison to that in wild-type cells. In addition, increased intracellular ROS accumulation appeared in the Trr1 shRNA knockdown cells compared to the RKO wild-type cells, in proportion to a relatively higher fraction of the DNA damage reporter protein phosphorylated histone 'γ-H2AX'. Notably, a neutral comet assay demonstrated that DNA double-strand breaks were highly induced in the Trr1-deficient cancer cells in the presence of MMC, presumably stimulating cancer cell death. Our results also revealed that MMC-induced apoptosis was associated with enhancement of oxidative damage to DNA. These results suggest that the specific knockdown of Trr1 expression via shRNA vector interference technology may be a potent molecular strategy by which to enhance the effectiveness of MMC-mediated killing in human colon cancer cells, through acceleration of double-strand DNA damage-oxidative stress as a trigger for apoptosis. This implies that Trr1 may be a prime target for enhancing the effectiveness of MMC chemotherapy in combination with specific RNA interference.