Colorectal cancer is the third leading cause of cancer related-death in the United States. Search for new alternatives to treat this type of cancer is necessary. In a previous report, auransterol from Penicillium aurantiacobrunneum showed cytotoxicity in HT-29 cancer cells. Thus, the goal of this study was to examine the potential cytotoxic mechanism of auransterol in HT-29 cells. Real-time cytotoxicity of auransterol was determined in HT-29 colon cancer cells, using the SRB assay. Loss of MTP, overproduction of ROS, cell cycle, cell migration, and caspase activity were analyzed. Western blot analysis was used to evaluate protein expression. Auransterol reduced cell proliferation rate in a time and concentration-dependent manner, with an IC₅₀ value > 100, 49.1 and 23.8 μM at 24, 48 and 72 h of treatment, respectively. After 24 h of treatment, 50 μM of auransterol induced loss of MTP, overproduction of ROS, increased caspase activity, induced cell cycle G₁ phase accumulation and inhibition of migration in HT-29 cells compared to control. These results were supported by protein upregulation of Cyt c, BAX, PARP-1, p21 and procaspase-3, and downregulation of Bcl-2 with no modifications in procaspase-7 and p53. The cytotoxic effect of auransterol in HT-29 colon cancer cells is mediated by mitochondrial apoptosis independent of p53 activation, cell cycle G₁ phase arrest, and inhibition of cell migration. This work encourages further preclinical and clinical studies of auransterol and suggests auransterol as a good candidate for colorectal cancer treatment.