Reactive oxygen species (ROS) are plant metabolic and signaling molecules involved in responses to various external stresses, but the existence of ROS receptors and how plants respond to ROS remain largely unknown. Here we report that the plasma membrane-localized phospholipase D δ (PLDδ) protein is crucial for sensing heat shock-induced ROS to initiate reorganization of guard cell microtubules in Arabidopsis cotyledons. Heat shock of wild-type Arabidopsis cotyledons stimulated ROS production which disrupted microtubule organization and induced stomatal closure, whereas this process was markedly impaired in pldδ mutants. Moreover, the wild-type PLDδ, but not the Arg-622-mutated PLDδ, complemented the pldδ phenotypes in heat shock-treated plants. ROS activated PLDδ by oxidizing cysteine residues, which was required for its functions in ROS-induced depolymerization of guard cell microtubules, stomatal closure, and plant thermotolerance. Additionally, lipid profiling reveals involvement of microtubules organization in the feedback-regulation of glycerolipid metabolism upon heat stress. Together, our findings highlight a potential mechanosensory role for PLDδ in regulating the dynamic organization of microtubules and stomatal movement, as part of the ROS-sensing pathway, during the response to external stresses.