Objectives:  The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non‐obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology. Materials and methods:  We have applied a cell‐sorting method, using collagen and magnetic activated cell separation to overcome obstacles, developing a collection system, and simple long‐term proliferation system, that yields large numbers of high‐purity SSCs from obstructive OA and NOA patients. Results:  SSCs derived from OA and NOA patients proliferated and maintained their characteristics for more than 12 passages (>6 months) in vitro. Moreover, the population of cells positive for the SSC‐specific markers GFRα‐1 and integrin α6, increased to more than 80% at passage 8. Conclusion:  These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.