The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Cζ, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH₂-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser⁹², Thr²⁰², and Thr²⁰⁵ of PP2Cζ. Phosphorylation of these Ser and Thr residues of PP2Cζ ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Cζ by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser⁹² but not at Thr²⁰² or Thr²⁰⁵ suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Cζ at Ser⁹² by stress-activated JNK attenuates its phosphatase activity in cells.