Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. While de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, whose activity is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes, and can regulate cofilin phosphorylation, we also asked if they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and MDCK) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM Kinase (LIMK) but not the Rac/PAK/LIMK pathway, because a) dominant negative (DN) Rho and DN-ROCK, but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; b) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; c) hyperosmolarity induced LIMK-2 phosphorylation, and d) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation. Next we examined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by siRNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.