Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of melatonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3-6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 μM) and stored for 1 month. Finally, the cell viability, colonization rate, expression of and gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and gene expression. It also diminished gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic.