Updated project metadata. The hyperosmotic stress response in budding yeast is a paradigm for cellular responses to physicochemical stimuli that is often used for modeling signal transduction pathways. Here, we describe the phosphatase PP2A-Cdc55 as a novel master regulator of hyperosmotic stress signaling. We show that its inhibition by the Greatwall kinase-Endosulfine signaling module at the onset of hyperosmotic stress is crucial for cellular survival with far-reaching consequences for the stress-regulated phospho-proteome. Indeed, this mechanism is required and sufficient to induce stress-specific phosphorylation patterns. This system operates in parallel and independently of the well-established Hog1 MAP kinase pathway, affecting up to 50% of the stress-induced S/T-P motifs. Many of these motifs appear to be direct substrates of PP2A-Cdc55. We exemplify the functional impact of stress-induced inhibition of PP2A-Cdc55 on the transcriptional regulation of stress-associated genes via the transcriptional regulators Rph1 and Gis1.