Allelopathy is a direct or indirect effect of one plant on another and occurs by releasing allelochemicals into the surrounding environment. When allelopathy inhibits the growth and development of receptor plants, it is considered a biotic stress called "allelochemical stress." Allelopathy is one of the successful mechanisms of alien plant invasions. Allelochemicals can enhance the production of reactive oxygen species (ROS) and increase membrane lipid peroxidation. Allelochemicals can also show obvious cytotoxicity and genotoxicity on receptor plants by inducing enzyme inactivation, DNA damage, and programmed cell death (PCD). Chenopodium ambrosioides L. (Mexican tea) is one of the aromatic herbaceous species of the Chenopodium genus, native to tropical Central and South America. It has recently become a major invasive plant in China, and has strong allelopathic effects on surrounding plants. This plant is threatening the ecological security of China in more than 20 provinces and cities by crowding out and suppressing native species. The guard cells are highly specialized cells, which are sensitive to the change of environment. These specialized cells could receive and integrate a great number of external and internal stimuli to accurately respond to various physiological requirements of the plant. Therefore, stomata are considered an optimal material for studying environmental stress. This study aimed to explore the mechanism of apoptosis and its signal regulation in guard cells induced by volatile oil from C. ambrosioides and by α-terpinene and cymene. Vicia faba L. (broad bean), which is widely planted in the areas invaded by C. ambrosioides, was chosen as the receptor plant. Epidermal strips of leaves were incubated in 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing volatile oil, α-terpinene and cymene for 30 minutes in light at 25℃ in an illumination incubator. After treatment, the epidermal strips were stained with acridine orange/ethidium bromide (AO/EB) to detect cell viability, and were also stained with histochemical localization to indicate intracellular reactive oxygen species (ROS), nitric oxide synthase (NOS) and calcium ion (Ca²⁺) levels. The results showed that volatile oil, α-terpinene, and cymene treatments resulted in a decrease in guard cell viability of broad bean leaves, and these effects were dose-dependent. Apoptosis features including nuclear malformation, karyorrhexis, and degradation were observed in guard cells under the volatile oil, α-terpinene, and cymene treatments. ROS, NOS and Ca²⁺ histochemical localization showed that the coloration of ROS, NOS and Ca²⁺ in guard cells deepened considerably under the treatments of volatile oil, α-terpinene and cymene. Meanwhile, the guard cell survival rates increased when epidermal strips were exposed to volatile oil, α-terpinene and cymene combined with different concentrations of ROS scavenger ascorbic acid (AsA), nitrite reductase inhibitor sodium azide (NaN₃), or Ca²⁺ chelator ethylene glycol tetraacetic acid (EGTA). All results suggested that ROS,NO, and Ca²⁺ participate in apoptosis signal pathways induced by volatile oil, α-terpinene, and cymene in V. fava stomata guard cells. Changes in Ca²⁺ levels regulated by ROS and NO may mediate programmed cell death induced by volatile oil, α-terpinene, and cymene.