In mammals, thioredoxin reductase 1 (Trr1) is of potential importance in defense against oxidative stress and regulation of redox homeostasis. Nevertheless, overexpression of this selenoprotein Trr1 is also manifested in several types of malignant cancers, leading to hypothesis that Trr1 might be a molecular candidate for anticancer drug treatment. An anthracycline antibiotic, doxorubicin (DOX), has been extensively applied in the chemotherapy of solid tumor tissues including colon. Cytotoxification of DOX is targeting on damage to DNA molecules via possible generation of reactive oxygen species (ROS) with consequent inhibition of macromolecule biosynthesis. At present work focusing on specific Trr1 knockdown using short hairpin RNA (shRNA) based viral vector, the important role of Trr1 on DOX resistance in human colon cancer RKO cell was deliberately investigated. Intriguingly, our finding presented that the stable Trr1 shRNA knockdown had higher sensitivity to DOX relative to the wild type. With respect to this point, accumulated intracellular ROS was notably stimulated in the Trr1 shRNA defect rather than the wild type. Apparently, it was also direct linkage between ROS level and extent of phosphorylated histone "gamma-H2AX", indicative of DNA damage. Strikingly, our important observation revealed that in presence of DOX Trr1 deficiency probably accelerated double strand breaks to DNA, provided evidence as neutral comet assay. Additionally, our data demonstrated that DOX-induced apoptosis was also correlated with increment of oxygen radicals damage to DNA. Taken together, it was worthy to conclude that specific reduction of Trr1 expression via shRNA based interference might be a considerable molecular approach to improve effectiveness of DOX-mediated killing in treatment of human colon cancer cells, through stimulation of oxidative insult. This implied that Trr1 might be a plausible target in cancer therapy.