This article reports the effect of surface agitation of culture medium on ultrasound-mediated gene transfection in vitro and its possible mechanisms. The possibility of active induction of bubbles without using contrast microbubbles for effective gene transfer was also demonstrated. Cultured HeLa cells mixed with green fluorescent protein plasmid were exposed to 1.0 MHz ultrasound in 24-well culture plates. Up to 26% transfection efficiency in the survival cell population was achieved in samples exposed to 0.44 MPa ultrasound pulses with the presence of surface agitation. Inertial cavitation and bubble generation were observed throughout the ultrasound exposure. When surface agitation was suppressed by covering the medium surface with a thin membrane, bubble generation and gene transfection were significantly suppressed. Interestingly, transfection efficiency could be partially resumed by adding a small amount of culture medium onto the covering membrane to rebuild the surface agitation and bubble generation. Pressure fluctuation and transient high-pressure loci were found in samples with surface agitation. Numerical simulations of bubble dynamics showed that transient high pressures above the inertial cavitation threshold could generate bubbles, which might be subsequently stabilized at lower pressures by rectified diffusion, and exert strong shear forces that might create transient pores on cell membranes to facilitate gene transfer.