In vitro assessment of cytotoxicity based on electrochemical impedance spectroscopy (EIS) needs more quantitative methods to analyze the alteration of cell morphology and motility, and hence the potential risk to human health. Here, we applied electric cell-substrate impedance sensing (ECIS) to evaluate dose-dependent responses of human umbilical vein endothelial cells exposed to cytochalasin B. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increments were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1 microM. Our results show that cytochalasin B causes a decrease of junctional resistance between cells, an increase of membrane capacitance, and the reduction in micromotion.