Technologies for rapid and high-throughput separation of rare cells from large populations of other types of cells have recently attracted much attention in the field of bioengineering. Among the various cell separation technologies proposed in the past, dielectrophoresis has shown particular promise because of its preciseness of manipulation and noninvasiveness to cells. However, one drawback of dielectrophoresis devices is that their application of high voltage generates Joule heat that exposes the cells within the device to high temperatures. To further explore this problem, this study investigated the temperature field in a previously developed cell separation device in detail. The temperature rise at the bottom of the microfluidic channel in the device was measured using a micro-LIF method. Moreover, the thermofluidic behavior of the cell separation device was numerically investigated by adopting a heat generation model that takes the electric-field-dependent heat generation term into account in the energy equation. Under the operating conditions of the previously developed cell separation device, the experimentally obtained temperature rise in the device was approximately 20 °C, and the numerical simulation results generally agreed well. Next, parametric calculations were performed with changes in the flow rate of the cell sample solution and the solution conductivity, and a temperature increase of more than 40 °C was predicted. The results demonstrated that an increase in temperature within the cell separation device may have a significant impact on the physiological functions of the cells, depending on the operating conditions of the device.