Pulsed electric field (PEF) processing has been developed and applied in food industry for several purposes. In this study we used PEF for increasing the intracellular trehalose content in Lactobacillus plantarum WCFS1. Our results indicated that it is possible to increase intracellular trehalose content in Lactobacillus plantarum WCFS1 to ~100 mM with 75% survival when applying a PEF treatment with an electric field strength of 7.5 kV/cm. Fluorescence staining of PEF-treated cells with propidium iodide (PI) and SYTO 9 showed that at 7.5 kV/cm only a small fraction (23%) of the cells had a permeated membrane by this PEF treatment, of which approximately half had an irreversible permeated membrane. Resealing of the pores in the membrane for PI uptake was very fast, in the order of seconds. These results indicate that PEF treatment is promising for increasing intracellular trehalose, but further optimization is required to increase the trehalose content in all cells.