Alantolactone, a methanol extract of Inula helenium, possesses anticancer properties in a number of cancer cell lines. However, its anticancer effect on human colorectal cancer cells and the underlying mechanisms remain to be elucidated. In the present study, the effects of alantolactone on cell viability and apoptosis in RKO human colon cancer cells were investigated. Alantolactone treatment of RKO cells was found to result in dose‑dependent inhibition of cell viability and induction of apoptosis, accompanied with the accumulation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential. In addition, these effects were blocked with N‑acetylcysteine, a specific ROS inhibitor. Western blotting indicated that exposure of RKO cells to alantolactone is associated with the downregulation of Bcl‑2, induction of Bax and activation of caspase‑3 and ‑9. These results indicated that a ROS‑mediated mitochondria‑dependent pathway is involved in alantolactone‑induced apoptosis. From these observations, it was hypothesized that alantolactone may be used for the treatment of human colon cancer.