Abstract: Ailanthone, a quassinoid isolated from Ailanthus altissima, is a promising candidate for natural herbicides. The herbicidal mechanism of action of ailanthone was preliminarily investigated in this study by the measurement of physiological indicators and germination test in a dish. Ailanthone had no effect on the capacity of seeds water uptake. Ailanthone strongly inhibited plant cells division with a mitotic index of 0.48% at the concentration of 1.00 μg/mL, as compared to 5.85% in the clear water control, and it could cause cell division malformation. Combined information such as enzymes activity assays of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) activity implied that the antioxidant capacity of plants could be improved by ailanthone. Reactive oxygen species (ROS) staining indicated that the outbreak of ROS could be induced with the concentration of 0.10 μg/mL ailanthone. The results of cell mortality showed that the presence of ailanthone could cause a certain percentage of cell inactivity. Ailanthone showed herbicidal activity by affecting multiple targets. Ailanthone could effectively inhibit plant cells division and the accumulation of ROS.