Provided are: a method for separating mammalian sperms, whereby it becomes possible to separate a large quantity of sperms within a short time and it also becomes possible to prevent the deteriorationin movement of the separated sperms; an artificial insemination method; and an in vitro fertilization method. In the method for separating mammalian sperms, the sperms are cultured in a culture medium containing a TLR7 ligand and are then separated into sperms floating in an upper layer in the culture medium and sperms precipitating in a lower layer in the culture medium. The artificial insemination method and the in vitro fertilization method are methods in which the artificial insemination and the in vitro fertilization in a non-human mammal are carried out, respectively, using sperms of the non-human mammal which are separated by a method for separating mammalian sperms.