Hyperosmotic shrinkage induces multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. Here we investigated the possible roles of ezrin/radixin/moesin (ERM) proteins in these events. Osmotic shrinkage of Ehrlich Lettre ascites cells elicited the formation of long microvillus-like protrusions, rapid translocation of endogenous ERM proteins and green fluorescent protein-tagged ezrin to the cortical region including these protrusions, and Thr567/564/558 (ezrin/radixin/moesin) phosphorylation of cortical ERM proteins. Reduced cell volume appeared to be the critical parameter in hypertonicity-induced ERM protein activation, whereas alterations in extracellular ionic strength or intracellular pH were not involved. A shrinkage-induced increase in the level of membrane-associated phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] appeared to play an important role in ERM protein activation, which was prevented after PtdIns(4,5)P2 depletion by expression of the synaptojanin-2 phosphatase domain. While expression of constitutively active RhoA increased basal ERM phosphorylation, the Rho-Rho kinase pathway did not appear to be involved in shrinkage-induced ERM protein phosphorylation, which was also unaffected by the inhibition or absence of Na+/H+ exchanger isoform (NHE1). Ezrin knockdown by small interfering RNA increased shrinkage-induced NHE1 activity, reduced basal and shrinkage-induced Rho activity, and attenuated the shrinkage-induced formation of microvillus-like protrusions. Hyperosmolarity-induced cell death was unaltered by ezrin knockdown or after phosphatidylinositol 3-kinase (PI3K) inhibition. In conclusion, ERM proteins are activated by osmotic shrinkage in a PtdIns(4,5)P2-dependent, NHE1-independent manner. This in turn mitigates the shrinkage-induced activation of NHE1, augments Rho activity, and may also contribute to F-actin rearrangement. In contrast, no evidence was found for the involvement of an NHE1-ezrin-PI3K-PKB pathway in counteracting shrinkage-induced cell death.