Disrupted apoptosis not only confers a survival advantage to cancer cells but also causes resistance to chemotherapies. Therefore, inducing cell apoptosis has become a promising strategy for anticancer treatment. Patrinia scabiosaefolia (PS) has long been used to clinically treat various types of malignancies including colorectal cancer (CRC). However, the precise mechanism of its tumoricidal activity remains largely unclear. Using a CRC mouse xenograft model and a human colon carcinoma cell line, HT-29, in the present study, we evaluated the antitumor activities of an ethanol extract of Patrinia scabiosaefolia (EEPS), and investigated the underlying molecular mechanisms. We found that EEPS inhibited CRC growth both in vivo and in vitro, without apparent adverse side-effects. Moreover, EEPS treatment promoted apoptosis in CRC tumor tissues and in HT-29 cells, suggesting that the inhibitory effect of EEPS on tumor growth was due to its pro-apoptotic activity. Furthermore, EEPS treatment inhibited the expression of anti-apoptotic Bcl-2 but enhanced pro-apoptotic Bax expression at both transcriptional and translational levels. Finally, EEPS induced the loss of mitochondrial membrane potential and activation of caspase-9 and -3 in HT-29 cells. Taken together, data in this study suggest that induction of cancer cell apoptosis via the mitochondrial-dependent pathway may be one of the mechanisms whereby PS exerts anticancer activity.