Tumor hypoxia contributes to the development of resistance to chemotherapeutic drugs in several human cancer cell lines. Atovaquone, an anti-malaria drug approved by the US Food and Drug Administration, has recently demonstrated anti-cancer effects in vitro and in vivo in several cancer models. To assess the potential of atovaquone as an anti-cancer agent under hypoxia in colorectal carcinoma, EpCAM+CD44+ colon cancer stem cells were isolated from HCT-116 human colon cancer cells through magnetic-activated cell sorting. The efficacy of atovaquone on cytotoxicity, tumorsphere formation, apoptosis, invasion and cell-cycle progression under hypoxic conditions were evaluated. MTS assays indicated that atovaquone inhibited the proliferation of EpCAM+CD44+ HCT-116 cells with a half-maximal inhibitory concentration of 15 µM. Atovaquone inhibited tumorsphere formation and cell proliferation by causing cell-cycle arrest in S-phase, which induced apoptosis of EpCAM+CD44+ HCT-116 cells, as detected by Annexin V-FITC/PI double staining assays, and caused mitochondrial membrane potential depolarization, as determined by a JC-1 staining assay. Reverse transcription-quantitative PCR demonstrated increased expression of Bax and downregulation of Bcl-2. Transwell invasion assays indicated that atovaquone inhibited the invasiveness of EpCAM+CD44+ HCT-116 cells under hypoxia, which was associated with upregulation of MMP-2 and -9 and increased expression of tissue inhibitor of MMPs (TIMP)-1. Taken together, atovaquone reduced the tumorsphere formation and invasion ability of EpCAM+CD44+ HCT-116 cells, at least in part by increasing the expression of TIMP-1 and downregulating the expression of MMP-2 and -9, as well as the cells' viability by inducing cell-cycle arrest in S-phase and induction of apoptosis via the Bcl-2/Bax pathway under hypoxic conditions. Further studies are warranted to explore the mechanisms of action of atovaquone as a promising anticancer agent in the treatment of colorectal carcinoma.