Juniperus chinensis is a well-known folk remedy in Korea, shown to exhibit anti-tumor, anti-bacterial, anti-fungal, abortifacient, anti-platelet, vasorelaxing, and anti-viral activities. In this study, we report the cytotoxic activity as well as the apoptotic mechanism of widdrol extracted from Juniperus chinensis against the human colon adenocarcinoma cell line HT29. Treatment of HT29 cells with various concentrations of widdrol inhibited growth and induced apoptosis in a dose-dependent manner, as determined by cell viability, chromatin condensation, and sub-G1 phase accumulation. Western blot analysis revealed that apoptosis of HT29 cells was associated with release of cytochrome C from mitochondria into the cytosol, activation of caspases-3, -8, and -9, and proteolytic cleavage of PARP. These results suggest that widdrol exhibits an anti-proliferative effect on HT29 cells via apoptosis and can be a potential candidate in the field of anti-cancer drug discovery.