Deferoxamine (DFO), an iron chelator, has numerous clinical applications for patients presenting with iron overload in regards to the improvement in the quality of life and overall survival. In addition, experimental iron chelators have demonstrated potent anticancer properties. The present study investigated the effects of DFO on TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in colon cancer cells and and the mechanism involved. The experimental results showed that DFO treatment inhibited TRAIL-mediated cancer cell apoptosis by increasing Akt activation and decreasing caspase activation in human colon cancer cells. Furthermore, DFO treatment induced autophagy flux, and chloroquine, an autophagy inhibitor, blocked DFO-mediated inhibition of TRAIL-induced apoptosis. The present study demonstrated that DFO inhibited TRAIL-mediated tumor cell death via the autophagy pathway, and the results suggest that potent anticancer agent, DFO, can be an inhibitor against antitumor therapy including TRAIL protein.