Increasing carbon dioxide (CO₂) levels in the atmosphere have caused global metabolic changes in diverse plant species. CO₂ is not only a carbon donor for photosynthesis but also an environmental signal that regulates stomatal movements and thereby controls plant-water relationships and carbon metabolism. However, the mechanism underlying CO₂ sensing in stomatal guard cells remains unclear. Here we report characterization of Arabidopsis RESISTANT TO HIGH CO₂ (RHC1), a MATE-type transporter that links elevated CO₂ concentration to repression of HT1, a protein kinase that negatively regulates CO₂-induced stomatal closing. We also show that HT1 phosphorylates and inactivates OST1, a kinase which is essential for the activation of the SLAC1 anion channel and stomatal closing. Combining genetic, biochemical and electrophysiological evidence, we reconstituted the molecular relay from CO₂ to SLAC1 activation, thus establishing a core pathway for CO₂ signalling in plant guard cells.