The Artemisia absinthium (AA), belongs to the Asteraceae family, is used as a therapeutic agent in traditional medicine in Iran. It is a rich source of biology-active compounds. However, the molecular mechanism of AA contributing to cell proliferation and apoptosis is still unknown. This study aims to assess the anticancer activity of the methanolic extract of A. absinthium (MEAA) against human colorectal cancer HCT-116 cell line. The cytotoxic effects of MEAA on HCT-116 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. The expression levels of BAX and BCL-2 in HCT-116 cell line were examined by qRT-PCR. Annexin V/PI-flow cytometry technique was used to detect the cell cycle and apoptosis. MMP was predicted by Rhodamine 123 staining, and caspase 3 activity was analyzed by ELISA. Western blot method was performed to detect the expression level of BAX, Bcl-2 and Caspase-3 proteins. The MTT test revealed MEAA reduced the viability of HCT-116 cells. The mRNA and protein levels of BAX increased, but those of BCL-2 decreased in MEAA-treated cells. MEAA also prompted cell cycle arrest and induced apoptosis. After adding MEAA, the protein level and activity of caspase 3 and MMP destruction significantly increased. MEAA predominantly prompted apoptosis in HCT-116 cells by activating the mitochondrial pathway.