Particle focusing in microfluidic devices is a necessary step in medical applications, such as detection, sorting, counting, and flow cytometry. This study proposes a microdevice that combines insulator-based and metal-electrode dielectrophoresis for the three-dimensional focusing of biological cells. Four insulating structures, which form an X pattern, are employed to confine the electric field in a conducting solution, thereby creating localized field minima in the microchannel. These electrodes, 56-μm-wide at the top and bottom surfaces, are connected to one electric pole of the power source. The electrodes connected to the opposite pole, which are at the sides of the microchannel, have one of three patterns: planar, dual-planar, or three-dimensional. Therefore, low-electric-field regions at the center of the microchannel are generated to restrain the viable HeLa cells with negative dielectrophoretic response. The array of insulating structures aforementioned is used to enhance the performance of confinement. According to numerical simulations, three-dimensional electrodes exhibit the best focusing performance, followed by dual-planar and planar electrodes. Experimental results reveal that increasing the strength of the applied electric field or decreasing the inlet flow rate significantly enhances focusing performance. The smallest width of focusing is 17 μm for an applied voltage and an inlet flow rate of 35 V and 0.5 μl/min, respectively. The effect of the inlet flow rate on focusing is insignificant for an applied voltage of 35 V. The proposed design retains the advantages of insulator-based dielectrophoresis with a relatively low required voltage. Additionally, complicated flow controls are unnecessary for the three-dimensional focusing of cells.