Mammalian cells are confronted with changes in extracellular osmolality at various sites, including the aqueous layer above the lung epithelium. Hypertonic shock induces the activation of mitogen-activated protein kinases and the expression of a defined set of genes, including aquaporins. We investigated upstream components of the response to hypertonicity in lung epithelial cells and found that before extracellular signal-regulated kinase activation and aquaporin synthesis, the membrane-bound prohormone neuregulin 1-β is cleaved and binds to human epidermal growth factor receptor 3 (HER3). The signaling is prevented by matrix metalloproteinase inhibition, inhibition of neuregulin 1-β binding to HER3, and inhibition of HER tyrosine kinase activity. Inhibition of HER activation interferes with the hypertonic induction of two different aquaporins in three distinct cell lines of mouse and human origin. We propose that ligand-dependent HER activation constitutes a generalized signaling principle in the mammalian hypertonic stress response relevant to aquaporin expression.