Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca²⁺](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca²⁺](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca²⁺](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca²⁺](cyt) oscillations. Copyright © 2012 Elsevier GmbH. All rights reserved.