Grazing, one of the primary utilization modes of grassland, is the main cause of grassland degradation. Historical overgrazing results in dwarf phenotype and decreased photosynthesis of perennial plants. However, it remains unknown what the mechanism underlying of this legacy effect is, and the role of stomata in the resulting decreased photosynthesis also remains unclear. To address these questions, differences in stomatal density, length and width on both adaxial and abaxial epidermis were compared between overgrazing and ungrazed Leymus chinensis offspring by using rhizome buds cultivated in a greenhouse, and the correlation between photosynthetic capacity and stomatal behavior was also investigated. Our results showed that historical grazing significantly impacted phenotype, photosynthesis and stomatal traits of L. chinensis. The offspring plants taken from overgrazed parents were dwarfed compared to those taken from ungrazed parents, and the photosynthesis and stomatal conductance of plants with a grazing history decreased by 28.6% and 21.3%, respectively. In addition, stomatal density and length on adaxial and abaxial leaf surfaces were significantly increased; however, stomatal width on abaxial leaf surfaces of overgrazed L. chinensis was significantly decreased compared with ungrazed individuals. Moreover, the expression patterns of eight genes related to stomatal regulation were tested: seven were down-regulated (2-18 times) and one was up-regulated (three times). Genes, involved in ABC transporter and receptor-like serine/threonine protein kinase were down-regulated. These results suggest that legacy effects of historical grazing affect the stomatal conductance by decreasing the stomatal width in progeny plants, which thus results in lower photosynthesis. Furthermore, changes of stomatal traits and function were regulated by the inhibition of ABC transporter and serine/threonine protein kinase. These findings are helpful for future exploration of the possible mechanisms underlying the response of grassland plants to long-term overgrazing. © 2020 Yin et al.