Myotubularins (MTMs) are lipid phosphoinositide 3-phosphate phosphatases and the product of their enzyme activity - phosphoinositide 5-phosphate (PtdIns5P) - functions as a signalling molecule in pathways involved in membrane dynamics and cell signalling. Two Arabidopsis genes, AtMTM1 and AtMTM2, encode enzymatically active phosphatases but although AtMTM1 deficiency results in increased tolerance to dehydration stress and a decrease in cellular PtdIns5P, the role of AtMTM2 is less clear, as it does not contribute to the PtdIns5P pool upon dehydration stress. Here we analysed the involvement of AtMTM1, AtMTM2 and PtdIns5P in the response of Arabidopsis seedlings to dehydration stress/ABA, and found that both AtMTM1 and AtMTM2 were involved but affected oppositely stomata movement and the accumulation of reactive oxygen species (ROS, e.g. H2O2). Acting as a secondary messenger in the ABA-induced ROS production in guard cells, PtdIns5P emerges as an evolutionarily conserved signalling molecule that calibrates cellular ROS under stress. We propose the biological relevance of the counteracting AtMTM1 and AtMTM2 activities is to balance the ABA-induced ROS accumulation and cellular homeostasis under dehydration stress.