This paper reports a new dielectrophoretic separation method of porcine oocytes for in vitro fertilization. Conventional manual selection of oocyte highly depends on the expert's experience and lacks universal standards for identifying the quality of oocyte. In this study, an electrode array chip with castellated shape was developed to evaluate dielectrophoretic velocities of oocytes, under applied bias conditions with an AC 3 V waveform at 1 MHz for 15 s. Based on different dielectrophoresis (DEP) response, the selected group of oocytes that moved showed a better developmental potential than the group of oocytes that stayed, representing a higher rate of blastocyst formation and a lower rate of polyspermic fertilization. In addition, the overall developmental potential of oocytes selected by the DEP device was comparable to that of oocytes selected by conventional manual method. These results demonstrate that the difference in dielectrophoretic velocity can be used to establish an objective criterion for the selection of oocytes. Consequently, this method will open the possibility to develop an automatic tool for oocyte selection, which would be helpful for assisted reproductive technologies such as transgenic and clonal animal production.