The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2. In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity. This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals.