In this study, distinguishing skin cancer cells (A431) and normal cells (HaCaT) was achieved using electrical impedance spectroscopy (EIS) with a novel developed device. The proliferation behaviors of the two cell types during a culture period of 5 days were characterized by the normalized impedance measured at 1465 Hz with simultaneous microscopic imaging for assistance. By fitting to the established equivalent circuits, A431 cells generated smaller resistance (R-c) values with smaller increasing variation, and comparable capacitance (C-c) values with similar decreasing variation compared with HaCaT cells. Moreover, C-c values were linearly correlated to the cell number. The results indicate that these two cell types can be distinguished with EIS based on the differences in the values and variation trends of R-c and C-c during the proliferation process in a real-time and label-free manner. Our work supplies a useful analytical approach for skin cancer cell research and may facilitate the early diagnosis of skin cancer.