We have proposed and developed a vibratory microinjection system (VMS) to facilitate gene transfer and to increase the efficiency in production of transgenic animals. The VMS injects transgene into a cell by vibrating an injection micropipette longitudinally at a frequency in the order of kilohertz. The vibrator in the second version of VMS consisted of one cylindrical stacked-type piezoelectric actuator and a housing, and provided any vibration frequency lower than 18 kHz. First of all, we found that the amplitudes at the level of the vibrator were perfectly proportional to the voltages applied to the vibrator. Then, we investigated both possible negative and positive effects of VMS. (1) Vibration may break transgene, but we could not find any fragmentation of a 3.5-kilobase-pair (kb) transgene even after an extremely long period of vibration at an extremely high injection pressure. (2) In a one-hour in vitro experiment, the vibratory microinjection ejected more than double the volume of transgene solution that the conventional (non-vibratory) microinjection did, indicating that the vibration increased the ejection speed by more than 100%. (3) Compared with the non-vibratory microinjection, we investigated the effects of VMS on pronuclear microinjection, in which a small amount of transgene is injected into one of two pronuclei in a zygote, and examined two vibration conditions: 5 kHz – 15 Vp-p and 10 kHz – 8 Vp-p. The vibratory microinjection pierced zygotes with significantly smaller cellular deformation, decreased the lethal events of a micropipette pulling out pronuclear components, and resulted in significantly better in vitro development to the blastocyst stage, although considerably higher death rates were also observed. The coexistence such as better embryonic development and higher death rate seemed contradictory, but the contradiction was probably derived from wide variations in amplitude among micropipettes. More specifically, we applied a fixed vibration condition to different micropipettes because we had confirmed that the amplitudes at the level of vibrator were stable. The micropipettes, however, could have been considerably different in vibration characteristic although they were commercially available products in accordance with industrial standard. Therefore, some micropipettes would have been effective, others ineffective or even injurious. In conclusion, the second version of VMS heralded a new approach to pronuclear microinjection because it showed several merits except for the higher death rates.