This study was designed to understand the growth characteristics of spermatogonial stem cells (SSCs) of cynomolgus monkeys (Macaca fascicularis)and establish an in vitro culture system. Unilateral testis from male cynomolgus monkey of juvenile stage was obtained by surgical operation, and testicular cells were collected by two-step enzymatic digestion method. Purified SSCs were cultured in a special medium in vitro and cultured SSCs were identified by alkaline phosphatase (AKP) dye. Meanwhile, clone formation rate was used to evaluate the effect of different feeder cells and different seeding densities on SSCs proliferation. The results showed that the culture medium consisted of DMEM supplemented with 10% FBS supported SSCs survival and proliferation in vitro. The typical clone clumps formed during 3-5 days and were positive for AKP dye. Judged from the observations 3 days and 7 days after culture, SSCs plated on Sertoli cells (SC) feeder layer performed better than on cynomolgus monkey skin fibroblast (CSF) cells feeder layer, with significant difference in clone formation rate (P<0.05), and 1×10⁵ cells/ml SSCs seeding density was proper for primary culture. This culture system provides convenience for the research of spermatogenesis mechanism and germ-line cells modification by gene targeting technology in nonhuman primates.