AbstractBacterial flagellin (flg22) induces rapid and permanent stomatal closure. However, its local and systemic as well as tissue‐ and cell‐specific effects are less understood. Our results show that flg22 induced local and systemic stomatal closure in intact tomato plants, which was regulated by reactive oxygen‐ and nitrogen species, and also affected the photosynthetic activity of guard cells but not of mesophyll cells. Interestingly, rapid and extensive local expression of Ethylene response factor 1 was observed after exposure to flg22, whereas the relative transcript levels of Defensin increased only after six hours, especially in systemic leaves. Following local and systemic ethylene emission already after one and six hours, jasmonic acid levels increased in the local leaves after six hours of flg22 treatment. Using immunohistochemical methods, significant defensin accumulation was found in the epidermis and stomata of flg22‐treated leaves and above them. Immunogold labelling revealed significant levels of defensins in the cell wall of the mesophyll parenchyma and guard cells. Furthermore, single cell qRT‐PCR confirmed that guard cells are able to synthesise defensins. It can be concluded that guard cells are not only involved in the first line of plant defense by regulating stomatal pore size, but can also defend themselves and the plant by producing and accumulating antimicrobial defensins where phytopathogens can penetrate.