Electric cell–substrate impedance sensing (ECIS) has been used as a real-time impedance-based method to quantify cell behavior in tissue culture. The method is capable of measuring both the resistance and capacitance of a cell-covered microelectrode at various AC frequencies. In this study, we demonstrate the application of high-frequency capacitance measurement (f = 40 or 64 kHz) for the sensitive detection of both the micromotion and wound-healing migration of human mesenchymal stem cells (hMSCs). Impedance measurements of cell-covered electrodes upon the challenge of various concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), from 0.1 to 30 μM, were conducted using ECIS. FCCP is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ATP production. By numerically analyzing the time-series capacitance data, a dose-dependent decrease in hMSC micromotion and wound-healing migration was observed, and the effect was significantly detected at levels as low as 0.1 μM. While most reported works with ECIS use the resistance/impedance time series, our results suggest the potential use of high-frequency capacitance time series for assessing migratory cell behavior such as micromotion and wound-healing migration.