Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca2+ channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca2+ channels activated by L-methionine (L-Met) at physiological concentrations and regulate stomatal apertures and plant growth. The glr3.1/3.5 mutations resulted in a lower cytosolic Ca2+ level, defective Ca2+-induced stomatal closure, and Ca2+-deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca2+ channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca2+ channels are distinct from previously characterized ROS-activated Ca2+ channels and act upstream of ROS, providing Ca2+ transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca2+ channels responsible for maintaining basal [Ca2+]cyt, play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture.