【Objective】The aim of this research was to optimize the cryopreserving conditions of porcine spermatogonial stem cells (PSSCs) and observe the changes of ultrastructure in PSSCs.【Method】The testicular cells of Landrace piglets aged from 1 day to 5 days were isolated by two-step enzymatic digestion method. The percoll discontinuous density gradients method was used to purify PSSCs. The transmission electron microscope (TEM) was used to observe the ultrastructure of PSSCs during different culturing periods in vitro. The effects of glycerine and dimethyl sulfoxide (DMSO) on PSSCs cryopreserving were investigated.【Result and conclusion】The results showed that the cell membrane of undifferentiated PSSCs was integrated and smoothed without pseudopodia, the cytoplasm was homogeneous and nucleus was clear. The pseudopodia were formed in differentiated PSSCs and the number of the mitochondria greatly increased. The appropriate cryopreserving medium for PSSCs is V(DMSO)∶V(DMEM/F12)∶V(FBS)∶V(100×Penicillin/Streptomycin solution) =10∶69∶20∶1.