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Zedoary turmeric oil (ZTO) is a volatile oil that is extracted from the dry rhizome of Curcuma zedoaria with a variety of biological activities, including anti-tumor activity. However, there is a lack of knowledge about the effect and mechanism of ZTO in human colon cancer cells. The aim of this study was to examine the potential efficacy of ZTO against human colon cancer cells (HCT116) and to uncover the molecular mechanisms of its anti-tumor effects. The anti-proliferative activity of ZTO was determined by the MTT assay, cell counts and colony formation assay. Senescent cells were detected using SA-β-Gal staining, while apoptosis and the CD44+ subpopulation were evaluated by flow cytometry. The expression levels of senescence- and apoptosis-related proteins were examined using western blotting. The results showed that treatment with ZTO significantly inhibited the growth of HCT116 cells and caused senescence and apoptosis in a dose- and time-dependent manner. Western blotting revealed that ZTO significantly increased the expression of senescence- and apoptosis-related proteins p16, p21, and p53 and the phosphorylation of ERK. Moreover, ZTO treatment reduced the cancer stem-like CD44 positive cell population. These findings suggest that ZTO inhibits human colon cancer cells by inducing senescence and apoptosis.