Abstract: Hu sheep is a valuable sheep breed in China, and semen cryopreservation of Hu sheep is important for sustainable development of the agri-food industry. This study aimed to evaluate the effect of thawing rate and antioxidants (procyanidins [PC] and mitoquinone [MitoQ]) on the quality and antioxidant enzyme activity of post-thaw sperm in Hu sheep. Our results showed that the highest sperm quality was obtained from the group thawed at 70°C for 5 seconds. Furthermore, addition of 150 nM MitoQ in the extender could enhance motility, integrity of the membrane and acrosome, and mitochondrial activity, whereas only sperm motility and membrane integrity were increased with 10 μg/mL of PC supplementation, compared with the control group. Meanwhile, both PC (10 μg/mL) and MitoQ (150 nM) supplementation increased the levels of superoxide dismutase and glutathione peroxidase and decreased the levels of reactive oxygen species and malondialdehyde. In conclusion, the optimal thawing protocol of semen cryopreservation in Hu sheep was 70°C for 5 seconds. MitoQ supplementation (150 mM) in the extender could improve sperm quality and reduce the level of oxidative stress in Hu sheep semen after cryopreservation. Further studies are needed to evaluate fertility of the post-thaw semen using MitoQ.