Our research to seek active compounds against human colorectal cancer from the root of Alkanna tinctoria (L.) Tausch led to the isolation of two naphthoquinones, alkannin (1) and angelylalkannin (2). The antiproliferative effects of the two compounds on human colon cancer cells HCT-116 and SW-480 were determined by the 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) method. Cell cycle profile and cell apoptosis were determined using flow cytometry. Both of the two compounds showed significant inhibitory effects on the cancer cells. For alkannin (1) and angelylalkannin (2), the median inhibitory concentration (IC50) values were 2.38 and 4.76 µ m for HCT-116 cells, while for SW-480 cells they were 4.53 and 7.03 µ m, respectively. The potential antiproliferative mechanisms were also explored. At concentrations between 1–10 µ m, both compounds arrested the cell cycle at the G1 phase and induced cell apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.