Cytochalasin B (CB) is a cell-permeable mycotoxin. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments, it inhibits cell movement and induces nuclear extrusion. In the present study, we investigated the anticancer activity of CB in HeLa human cervical carcinoma cells. CB showed significant cytotoxicity, with an IC50 of 7.9 µM, in a WST-8 assay and significantly inhibited cell proliferation. Furthermore, results from Annexin V-FITC/propidium iodide double-staining indicated that CB induced early apoptosis of HeLa cells in a time-dependent manner. The cells exhibited apoptotic morphology, including cell shrinkage and nuclear condensation. CB induced cell cycle arrest at the S phase. We also observed inhibition of DNA replication in a [3H]-thymidine incorporation assay. Furthermore, CB induced a time-dependent increase in reactive oxygen species and a decrease in mitochondrial membrane potential. Western blot analysis showed an increase in levels of mitochondrial factors Bax and Bcl-2, which was followed by activation of caspase-9 and -3. These results suggested that CB induced apoptosis via a mitochondrial-dependent pathway in HeLa cells.