Surface plasmon resonance analysis and molecular docking study revealed that costunolide directly interacted with TrxR1 via its lactone oxygen atom with Gln-494 of TrxR1 and inhibited the activity of TrxR1, thereby increasing the production of ROS and inducing ROS-dependent ER stress and apoptosis in colon cancer cells (HCT-116, SW-620, and HT-29 cells). This evidence concerns the gene TXNRD1 and malignant colon neoplasm.