Root border cells (RBCs) are a group of special cells that develops from root cap meristems and aggregates around the root cap during the development of plant root tips. The border cell-extracellular traps are formed by root border cells and their secreted substances. This structure provides a protective barrier between the root and the soil, and thus protects the root from adverse factors, including allelochemicals. Extracellular trapping nets are extremely important for root growth and development. An alien invasive plant, C. ambrosioides, has a strongly allelopathic effect on its surrounding plants. It can significantly inhibit the growth of roots and even the whole plant by affecting the cellular structure, physiological and biochemical process, and gene expression of receptor plants to suppress and exclude other plants. To further explore how the allelopathy of C. ambrosioides interferes with the defense function of the receptor plants, this study used the root border cells of V. faba, which is widely planted in the invasive areas of C. ambrosioides in southwestern China, as the experimental receptor. Under the treatments of volatile oil from C. ambrosioides and its main components (p-cymenin and α-terpene), the adhesive layer thickness, cell viability, intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels in the root border cells of V. faba were tested by using microscopy and spectrophotometry. The effects of ROS and NO on cell activity and the type of cell death of the root border cells were evaluated by applying ROS scavenging agent (ascorbic acid, AsA), nitrate reductase inhibitor (NaN₃), and pan-Caspase inhibitor (Z-VAD-FMK). The results showed that the adhesive layer thickness of RBCs increased, the cell activity decreased, and the levels of ROS and NO in the root border cells increased in exposure to the volatile oil, p-cymenin and α-terpenene, which showed a concentration-dependent effect. It was found that there was a significantly positive correlation among root border cell death rate, ROS and NO levels (P < 0.05) by using the heat map analysis of the related parameters of root border cells. The ROS scavenger (AsA), NaN₃, and Z-VAD-FMK could effectively alleviate the lethal effects of the volatile oil, p-cymenin and α-terpene on root border cells of V. faba, which indicated that ROS and NO induced Caspase-dependence apoptosis of root border cells. These results suggested that the volatile allelochemicals from C. ambrosioides induced root border cell death by increasing the levels of ROS and NO. Therefore, the allelopathy C. ambrosioides caused the protective dysfunction of the receptor plant by removing the protective barrier, and inhibited growth of roots of the receptor plant.