Intracytoplasmic sperm injection (ICSI) has been successfully achieved in mice and rats using a piezo-driven injection pipette. More than 30% of ICSI oocytes are capable of developing to full-term when the isolated sperm heads are microinjected. The ICSI technique has been applied not only to rescue infertile male strains, but also to produce transgenic rodents. ICSI-mediated DNA transfer, which mixes sperm heads and exogenous DNA solution and co-injects them into ooplasm, was as effective as conventional pronuclear DNA microinjection. The production efficiency of transgenic founders by ICSI-mediated DNA transfer was comparable between mice and rats, while the optimal DNA concentration for 1-min exposure was lower in rats than in mice. The production efficiency was improved when the membrane structure of sperm heads was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. Exogenous DNAs with various chain lengths have been stably integrated into rodent genomes of various genetic backgrounds using this method. ICSI-mediated DNA transfer in which preparation of pronuclear-stage fertilized zygotes is not required would be alternative to conventional pronuclear DNA microinjection.