Although normal human keratinocytes are known to migrate toward the cathode in a direct current (DC) electric field, other effects of the electric stimulation on keratinocyte activities are still unclear. We have investigated the keratinocyte differentiation under monodirectional pulsed electric stimulation which reduces the electrothermal and electrochemical hazards of a DC application. When cultured keratinocytes were exposed to the electric field of 3 V (ca. 100 mV/mm) or 5 V (ca. 166 mV/mm) at a frequency of 4,800 Hz for 5 min a day for 5 days, cell growth under the 5-V stimulation was significantly suppressed as compared with the control culture. Expression of mRNAs encoding keratinocyte differentiation markers such as keratin 10, involucrin, transglutaminase 1, and filaggrin was significantly increased in response to the 5-V stimulation, while the 3-V stimulation induced no significant change. After the 5-V stimulation, enhanced immunofluorescent stainings of involucrin and filaggrin were observed. These results indicate that monodirectional pulsed electric stimulation induces the keratinocyte differentiation with growth arrest.