Objective: To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells. Methods: Tissues of neonate NMRI male mice testes were used for harvesting spermatogonial stem cells and Sertoli cells. After cell harvest, flow cytometry using promyelocytic leukemia zinc-finger (PLZF) protein antibody was used to assess the purity of the cells. The isolated testicular cells were cultured in the absence (the control group) or presence of soft agar-coated dishes (the experimental group) supplemented with leukemia inhibitory factor and glia cell line–derived neurotrophic factor for two weeks. Alkaline phosphatase activity was assessed in the colonies formed after two weeks of culture by alkaline phosphatase staining. On day 14 of culture, the expression levels of DNA-binding protein inhibitor (ID-4) and PLZF genes in the undifferentiated cells were evaluated by the detection of PLZF protein antibody using real-time PCR and immunocytochemistry techniques. The number and diameter of the colonies of spermatogonial stem cells were assessed by ImageJ software. Results: In the experimental group, the number and the diameter of colonies significantly increased as compared with those in the control group (P<0.05, P<0.01, respectively). In addition, the level of expression of ID-4 and PLZF genes in the undifferentiated cells significantly increased in the experimental group as compared with the control group (P<0.01). However, the expression of tyrosine-protein kinase kit (c-kit) gene in differentiated cells decreased in the experimental group as compared with the control group, but there was no significant difference between the two groups. Conclusions: Spermatogonial stem cells can be efficiently proliferated by culturing the stem cells in soft agar-coated dishes. The new protocol used in this study can be a valuable method for future studies.