High recurrence and metastatic behavior patterns are the most important reasons for the failure of treatment strategies in patients with colon cancer. Cancer stem cells (CSCs), which are considered root of cancer, are thought to be associated with therapy resistance, relapse, and metastasis, and, therefore, targeting CSCs rather than the bulk population may be an effective approach. In cancer studies, there is an increasing interest in close friendship between epithelial-mesenchymal transition (EMT) and CSCs. Triptolide (TPL) isolated from Chinese herb Tripterygium wilfordii has important effects on the prevention of migration and metastasis as well as cytotoxic effect against cancer cells. The potential lethal efficacy of TPL on CSCs that is highly resistant to the drug is an unsolved mystery. Fundamentally, the present study basically aims to find answers to two questions: (a) is it possible to target colon CSCs with TPL? and (b) what are the mechanisms underlying TPL's potential to eliminate CSCs? Cytotoxic effects of TPL on CSCs were evaluated by WST-1 and Muse count and viability assays. Apoptosis assay and cell-cycle analysis were performed to investigate the inhibitory effect of TPL. Moreover, the effects of TPL on spheroid formation capacity, migration, and EMT processes, which are associated with CSC phenotype, were also investigated. The results revealed that TPL triggered cell death and apoptosis and altered cell cycle distribution. Moreover, TPL significantly reduced the snail slug and twist expressions associated with EMT. TPL has been shown to be effective in colon CSCs by in vitro experiments, and it might be a highly effective agent against colon cancer has been implicated in need of supporting in vivo and clinical studies.