The chapter talks about the concept of semen freezing. Cryopreservation of human semen is stabilizing the human spermatozoa by cryoprotectant agent at cryogenic temperature (–196°C) using liquid nitrogen. This is routine procedure used for intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in assisted reproduction technology (ART) laboratories. Slow freezing and vitrification are two commonly practiced cryopreservation methods. Under the cryopreserved conditions, all biological and biochemical processes are arrested preventing from cell death and DNA degradation. Cryopreservation of human semen is indicated for storage of sperm from ejaculate for IUI, IVF or ICSI, preservation of sperm from ejaculate, sperm donation, compromised fertility, and storage of testicular sperm for ICSI. The main role of cryoprotectant is to prevent crystallization of water to ice by locking molecules in place and forming amorphous solid. Commonly used cryoprotectant agents are glycerol, dimethyl sulfoxide, ethanediol, propanediol, etc.