Cryopreservation of spermatogonial stem cells (SSCs) is an advantageous method to restore fertility in young boys' cancer survivors. Recently, for reduction of SSCs cryoinjering various antioxidants have been used. The purpose of this study was to evaluate antioxidant effects of melatonin in frozen-thawed SSCs. Spermatogonial stem cells were isolated from testes of neonate mice (3-6 days old) and their purities were measured by flow cytometry with GFRα1. After culturing, the cells were frozen in two groups 1control and 2-melatonin (100μM) for one month. Finally, the cell viability, colonization rate, intracellular ROS were evaluated after freezing thawing. Melatonin increased the viability, colonization of SSCs, and decreased intracellular ROS. The results of this study show that melatonin with antioxidant effects as a good option can be used for freezing and long-term storage of cells and infertility treatment in the clinic.