Starting with an extract derived from the root of Cynanchum paniculatum Kitagawa (Asclepiadaceae) that was active in the process of inhibiting the growth of human cancer cells in culture, a phenanthroindolizidine alkaloid antofine was isolated and identified as an active principle (IC50 = 7.0 ± 0.2 ng/ml for A549, human lung cancer cells; IC50 = 8.6 ± 0.3 ng/ml for Co12, human colon cancer cells). Prompted by the high potency of cancer cell growth inhibition, additional action mechanism studies were performed with antofine. Utilizing cultured Co12 cells as a model, antofine induced arrest in the G2/M phase of the cell cycle after 48 h of incubation. With wash-out experiment, colony formation was also inhibited in a dose-dependent manner. These data suggest the potential of antofine to serve as a cancer chemotherapeutic agent by virtue of arresting the cell cycle.