A new and effective method to produce transgenic animals was established. Without a surgical incision, the recombinant plasmid containing green fluorescence protein (GFP) cDNA was repeatedly injected into male mouse testis at multi-sites. After few weeks of the final injection, the injected male was mated with normal oestrus female to produce transgenic mice. The presence of the GFP cDNA in F₁ transgenic individuals were detected by polymerase chain reaction and Southern blot hybridization, which showed that the transgenic rate of mouse F₁ offspring was 41%. The transferred gene was integrated into the host genome and could be transmitted to its offspring. When the positive F₁ individuals were mated with the wild type ICR mice, the F₂ individuals had a transgenic rate of 37%. The results indicate that the high efficiency of gene transfer and the limited number of manipulations make the method suitable for creating a large number of transgenic animals, especially, for producing domestic animals.