Intracytoplasmic sperm injection (ICSI) is the injection of a single spermatozoon directly into the cytoplasm of an oocyte using an injection pipette. Spermatozoa that are used for ICSI do not need to be motile or alive. Indeed, for mouse ICSI, the sperm tail is typically broken off before injection into the oocyte, resulting essentially in a nuclear transfer. The protocol describes creation of the injection pipettes, preparation of oocytes and spermatozoa, and steps of the ICSI procedure. The technique can be used to create both hybrid and inbred mice, as well as transgenic animals.