The purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium containing 200 mM trehalose, 40 µg/mL Vit C, and a combination of both for 3 weeks. Cell viability, colony number and diameter and mRNA levels of Bax, and Bcl-2 genes were evaluated before and after cryopreservation with quantitative real-time PCR. The results showed that cells cryopreserved in freezing medium containing 200 mM of trehalose plus 40 µg/mL Vit C had considerably greater cell viability than the control group (P<0.0001). Up to the 3rd week of cell culture, supplementation of freezing medium with 200 mM trehalose resulted in significantly lower colonies diameters than in the control group. No significant differences were observed during the 1st to 2nd weeks in colony diameter and number of colonies between cells cryopreserved in the freezing medium containing either Vit C or trehalose compared with the control groups. Following thawing, the mRNA level of Bax in the Vit C + trehalose group was drastically lower than in those treated with trehalose or Vit C only (P<0.0001). High expression of Bcl-2 in the 40 µg/mL Vit C group was recorded in the thawed cells compared to the control group (P<0.0001). These findings indicate that the concomitant use of antioxidants and sugar in the freezing medium can improve the quality and viabi­lity of SSCs after freezing via different mechanisms. Further studies are needed to clarify apoptosis and cell biomarkers in SSCs during freezing and thawing.