Spermatogonial stem cells (SSC) undergo self-renewal division and generate spermatogenesis to produce mature spermatozoa. Very recently in some species, include rodent and farm animals, SSC has been isolated and cultured in vitro. In this study, we analysed SSC marker of both 5 days old and pubertal pig testis by histological method. In 5day pig testis, prior to set of spermatogenic differentiation, the seminiferous tubules contain a relatively large number of SSCs than in pubertal pig testis. Then putative pig SSCs were successfully isolated from 5 day pig testis, and cultured long term using CD34 positive cell culture media contained GDNF, bFGF, LIF and EGF. The SSC colonies were appeared at 3 days after cells were seed. The SSC colonies were alkaline phosphatase positive and strongly expressed PGP 9.5, PLZF and DBA, but not expressed GATA4 and LH receptors. The SSC colonies were stably proliferated in GDNF, bFGF, LIF and EGF contained CD34 positive cell culture media up to 60 days. This study will be facilitated to study of in vitro and ex vivo spermatogenesis and of production of transgenic pigs using sperm vector.