Provided are a method for separating mammalian sperm, by which even a large amount of sperm can be separated in a short time and decrease in motility of the separated sperm can be prevented； and an artificial insemination method, and an in vitro fertilization method. The method for separating mammalian sperm includes incubating the sperm in a medium containing a TLR7 ligand, and separating them into sperm floating in the upper layer and sperm sinking in the lower layer of the medium. The artificial insemination and in vitro fertilization methods include artificial insemination or in vitro fertilization for a non-human mammal using sperm of the non-human mammal separated by the method for separating mammalian sperm.
