Intermediate filaments (IFs) play a key role in the control of cell structure and morphology, cell mechano‐responses, migration, proliferation, and apoptosis. However, the mechanisms regulating IFs organization in motile adhesive cells under certain physical/pathological conditions remain to be fully understood. In this study, we found hypo‐osmotic–induced stress results in a dramatic but reversible rearrangement of the IF network. Vimentin and nestin IFs are partially depolymerized as they are redistributed throughout the cell cytoplasm after hypo‐osmotic shock. This spreading of the IFs requires an intact microtubule network and the motor protein associated transportation. Both nocodazole treatment and depletion of kinesin‐1 (KIF5B) block the hypo‐osmotic shock–induced rearrangement of IFs showing that the dynamic behavior of IFs largely depends on microtubules and kinesin‐dependent transport. Moreover, we show that cell survival rates are dramatically decreased in response to hypo‐osmotic shock, which was more severe by vimentin IFs depletion, indicating its contribution to osmotic endurance. Collectively, these results reveal a critical role of vimentin IFs under hypotonic stress and provide evidence that IFs are important for the defense mechanisms during the osmotic challenge.