The invention discloses an establishment method for differentiating swine germline stem cells into sperms in vitro. The establishment method includes the steps of subjecting piglet testis tissues to isolation culture to obtain primary piglet testis tissue fibroblasts and germline stem cells, culturing the testis tissue fibroblasts for 15 days by means of a Niche culture medium for inducing in-vitro differentiation of sperms to obtain a Niche feeder layer for inducing in-vitro differentiation of sperms, and inoculating the germline stem cells to the Niche feeder layer to culture for 27-30 days, thereby obtaining haploid sperm-like cells in vitro by cytobiology analysis. The establishment method is easy to operate and good in repeatability, is an important breakthrough in research on swine mGSCs(male germline stem cells) in-vitro differentiation, provides an important basic technology for researches on swine embryonic in-vitro operation and transgenic breeding, and meanwhile, has high reference value in research on in-vitro differentiation of sperms of other species and assisted reproduction of human beings.