The aim of the current study was to evaluate the effects of cysteine and ergothioneine on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities. Semen samples from 5 mature Merino rams were used in the study. Semen samples, which were diluted with a Tris-based extender containing l-Cysteine and l-(+)-Ergothioneine and no antioxidant (control), were cooled to 5°C and frozen in 0.25. ml French straws. Frozen straws were then thawed individually at 37°C for 20. s in a water bath for evaluation.Ergothioneine at doses of 2 and 4. mM increased percentages of subjective motility, VSL and VCL, compared to controls following the freeze-thawing (P<0.001). Ergothioneine at three different doses led to higher rates of progressive motility and VAP, compared to control groups (P<0.001). Cysteine and ergothioneine at three doses provided the higher rates of ALH, in comparison to no antioxidant group (P<0.001). As regards CASA motility, supplementation with antioxidants did not provide any significant difference on the percentage of post-thaw sperm CASA motilities, in comparison to the control. In regards of sperm membrane integrity, only cysteine 1. mM provided a greater protective effect, compared to control (P<0.001). Percentages of sperm with high mitochondrial activity were dramatically increased with cysteine at doses of 1 and 2. mM, compared to control (P<0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1. mM compared to control group (P<0.001). Cysteine at doses of 2 and 4. mM showed a tendency of increased activities of CAT when compared to control. But these increases were not statistically significant. Supplementation with antioxidants did not significantly affect activities of SOD and GPx. Findings of this study showed that ergothioneine supplementation in semen extenders, was of greater benefit to motility and motion characteristics of frozen-thawed ram sperm. © 2011.