Current electrotransfection protocols are well-established for decades and, as a rule, employ long micro-millisecond range electric field pulses to facilitate DNA transfer while application of nanosecond range pulses is limited. The purpose of this paper is to show that the transfection using ultrashort pulses is possible by regulating the pulse repetition frequency. We have used 200 ns pulses (10-18 kV/cm) in bursts of ten with varied repetition frequency (1 Hz-1 MHz). The Chinese Hamster Ovary (CHO) cells were used as a cell model. Experiments were performed using green fluorescent protein (GFP) and luciferase (LUC) coding plasmids. Transfection expression levels were evaluated using flow cytometry or luminometer. It was shown that with the increase of frequency from 100 kHz to 1 MHz, the transfection expression levels increased up to 17% with minimal decrease in cell viability. The LUC coding plasmid was transferred more efficiently using high frequency bursts compared to single pulses of equivalent energy. The first proof of concept for frequency-controlled nanosecond electrotransfection was shown, which can find application as a new non-viral gene delivery method.