Freeze-drying spermatozoa is the ultimate method for the maintenance of animal strains, in that the gametes can be preserved for a long time in a refrigerator at 4 °C. Furthermore, it is possible to realize easy and safe transportation of spermatozoa at an ambient temperature that requires neither liquid nitrogen nor dry ice. Freeze-drying spermatozoa has been established as a new method for storing genetic resources instead of cryopreservation using liquid nitrogen. This chapter introduces our latest protocols for freeze-drying of mouse and rat spermatozoa, and the anticipated results of the fertilizing ability of these gametes following long-term preservation or transportation.