When cells are challenged by hyperosmotic stress, one of the crucial adaptive responses is the expression of osmoprotective genes that are responsible for raising the intracellular level of compatible osmolytes such as sorbitol, betaine, and myo-inositol. This is achieved by the activation of the transcription factor called OREBP (also known as TonEBP or NFAT5) that specifically binds to the osmotic response element (ORE) or tonicity-responsive enhancer that enhances the transcription of these genes. Here we show that p38, a subgroup of the mitogen-activated kinases activated by hypertonic stress, and Fyn, a shrinkage-activated tyrosine kinase, are both involved in the hypertonic activation of OREBP/TonEBP. Inhibition of p38 by SB203580 or by the dominant negative p38 mutant partially blocked the hypertonic induction of ORE reporter (reporter gene regulated by ORE). Similarly, hypertonic activation of ORE reporter was partially blocked by pharmacological inhibition of Fyn or by a dominant negative Fyn and was attenuated in Fyn-deficient cells. Importantly, inhibiting p38 in Fyn-deficient cells almost completely abolished the hypertonic induction of ORE reporter activity, indicating that p38 and Fyn are the major signaling pathways for the hypertonic activation of OREBP/TonEBP. Further we show that the transactivation domain of OREBP/TonEBP is the target of p38- and Fyn-mediated hypertonic activation. These results indicate a dual control in regulating the expression of the osmoprotective genes in mammalian cells.