Effects of PEF on permeabilization of calcein-filled large unilamellar vesicles (LUV) with average diameter of 1705.64 ± 55.91 nm and small unilamellar vesicles (SUV) with average diameter of 213.06 ± 5.50 nm, suspended in an isosmotic sucrose solution (150 mM) were investigated. The suspensions of vesicles were exposed to PEF at various electric field strengths in the interval of 0–50 kV/cm and different treatment times ranging from 0 to 4.8 ms. The intensity of permeabilization induced by PEF was analyzed in terms of the changes of fluorescence intensity of the suspension solution and the average diameter of vesicles, due to the leakage of self-quench calcein encapsulated in vesicles. Results indicated that the permeability of LUV was increased with increasing intensity of electric field (>10 kV/cm) and treatment time, and the release percentage of calcein (R value) was increased by 57.98% (from 0.46% to 58.44%) with the increase of the electric field strength (from 10 to 50 kV/cm) at 4.8 ms. However, the R value was only increased by 14.62% (from 43.82% to 58.44%) with the increase of the treatment time (from 1.2 to 4.8 ms) at 50 kV/cm. Average diameter of LUV was sharply decreased from 1705.64 ± 55.91 to 465.60 ± 37.12 nm with the maximum value at 50 kV/cm and 4.8 ms. Results also showed that shrinkage or rupture of LUV happened, as confirmed by the change of size distribution of LUV after treatment by PEF. However, as for SUV, the changes of the permeability of vesicles and average diameter were not observed. In addition, it was demonstrated that the magnitude of the induced transmembrane potential was proportional to the radius of the vesicle, and larger vesicles could be polarized or ruptured at relatively lower field strength than smaller vesicles.