Internalization of hydrophilic molecules into yeast cytosol is required for different applications such as cell transformation or preservation of water soluble components by bioencapsulation. However, these molecules are not able to cross the plasma membrane and strategies have to be developed. Recent works revealed that osmotic perturbations could induce non-lethal transient permeabilization of the plasma membrane. In this work, we endeavored to clarify the phenomenon of permeabilization during rehydration after a mild hyperosmotic perturbation in order to evaluate the possibility of hydrophilic molecule internalization in yeast by this treatment. Rehydration step is particularly interesting because the large entry of water into the cells could help the internalization of molecules. The internalization of a fluorescent molecule [fluorescein isothiocyanate Dextran (FITC-Dextran), 20 kDa], added during the rehydration after a sublethal hyperosmotic treatment, was studied in Saccharomyces cerevisiae yeast cells. The internalization kinetic and the localization of the fluorescent molecules were studied by flow cytometry and fluorescence confocal microscopy. Our results show that the rehydration leads to the rapid internalization of FITC-Dextran due to a transient plasma membrane permeabilization. Thus, osmoporation, i.e. plasma membrane poration by modifications of osmotic pressure of the extracellular medium, could be a new and simple way to deliver molecules of particular interest into yeasts.