Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space costs, as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve chromosomal integrity and oocyte-activating factor(s) in rodent and mammalian species at 4 °C for several years and at ambient temperature for up to 1 year depending on species, which permits shipping freeze-dried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm first and then presents a simple freeze-drying protocol.