The role of actin fibers in cellular responses to external electric pulses is not clear yet. In this study, we utilized the blocker of actin polymerization, cytochalasin D (cytoD), and investigated its effects on the electropore generation. Eight 100 mu s electric pulses of sub-kilovolt per centimeter voltage with 100 ms intervals were applied to adhered cells in vitro, and the membrane permeability was quantified using membrane-impermeable propidium iodide (PI) dye. With cytoD application, the transfer of PI dye decreased significantly in all the applied voltages. At the same time, the roughness of cells increased, the membrane stiffness decreased, and the transmembrane resting potential decreased. Our result supports that actin fibers have clear effects on electroporation through modulating membrane properties including transmembrane resting potential.