Objective To investigate the regulation of expression of H19 and A-myb by glial cell line-derived neurotrophic factor (GDNF), and explore the molecular mechanism of self-renewal of spermatogonial stem cells regulated by GDNF. Methods Spermatogonial stem cells were isolated, identified and cultured from testes of 6-day-old mice with mixed enzyme digestion and differential plating procedure. Spermatogonial stem cells and spermatogonial stem cells pretreated with LY294002, inhibitor of phosphotylinosital 3 kinase/protein kinase B(PI3K/Akt), were stimulated with GDNF, and the expression of H19 and A-myb in spermatogonial stem cells was detected by RT-PCR. Results Spermatogonial stem cells were successfully isolated and cultured. The expression of H19 and A-myb was upregulated in spermatogonial stem cells after stimulation with GDNF, while there was no significant change in the expression of H19 and A-myb in spermatogonial stem cells after pretreatment with LY294002. Conclusion GDNF can upregulate the expression of H19 and A-myb via PI3K/AKT signal pathway in cultured mouse spermatogonial stem cells.