Stomatal pores evolved more than 410 million years ago 1, 2 and allowed vascular plants to regulate transpirational water loss during the uptake of CO2 for photosynthesis 3. Here, we show that stomata on the sporophytes of the moss Physcomitrella patens 2 respond to environmental signals in a similar way to those of flowering plants 4 and that a homolog of a key signaling component in the vascular plant drought hormone abscisic acid (ABA) response 5 is involved in stomatal control in mosses. Cross-species complementation experiments reveal that the stomatal ABA response of a flowering plant (Arabidopsis thaliana) mutant, lacking the ABA-regulatory protein kinase OPEN STOMATA 1 (OST1) 6, is rescued by substitution with the moss P. patens homolog, PpOST1-1, which evolved more than 400 million years earlier. We further demonstrate through the targeted knockout of the PpOST1-1 gene in P. patens that its role in guard cell closure is conserved, with stomata of mutant mosses exhibiting a significantly attenuated ABA response. Our analyses indicate that core regulatory components involved in guard cell ABA signaling of flowering plants are operational in mosses and likely originated in the last common ancestor of these lineages more than 400 million years ago 7, prior to the evolution of ferns 8, 9. ► Mosses regulate stomatal apertures in a similar way to later-evolving flowering plants ► Moss stomatal apertures respond to light, CO2 concentration, and stress hormone ABA ► An OST1-like ABA signaling kinase is involved in moss stomatal aperture control ► Stomatal ABA signaling originated in a common ancestor more than 400 million years ago