The study presents the proof of concept for a possibility to achieve a better electroporation in the MHz pulse repetition frequency (PRF) region compared to the conventional low frequency protocols. The 200 ns × 10 pulses bursts of 10–14 kV/cm have been used to permeabilize Chinese hamster ovary (CHO) cells in a wide range (1 Hz–1 MHz) of PRF. The permeabilization efficiency was evaluated using fluorescent dye assay (propidium iodide) and flow cytometry. It was determined that a threshold PRF exists when the relaxation of the cell transmembrane potential is longer than the delay between the consequent pulses, which results in accumulation of the charge on the membrane. For the CHO cells and 0.1 S/m electroporation medium, this phenomenon is detectable in the 0.5–1 MHz range. It was shown that the PRF is an important parameter that could be used for flexible control of electroporation efficiency in the high frequency range.