The objective of this study was to examine the different concentrations of antipain and trehalose combination on post-thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A0 Tre0: antipain 0 μM and trehalose 0 mM (Control); A10 Tre0; A50 Tre0; A0 Tre30; A0 Tre60; A10 Tre60; A10 Tre30; A50 Tre30 and A50 Tre60). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 ml straws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A10 Tre0, A0 Tre30 and A10 Tre60 significantly (p < 0.05) higher as compared to the control (A10 Tre0). A10 Tre60 (47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility, VAP, VSL, VCL, ALH, BCF, STR and LIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A10 Tre0, A50 Tre0, A0 Tre30 and A10 Tre60, compared to control. Addition of A10 Tre0, A50 Tre0 and A10 Tre60 to extenders improved the percentages of sperm abnormality, compared to the controls. A10 Tre60 (67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A10 Tre0, A0 Tre60 and A10 Tre60 improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A50 Tre60 group. In conclusion, when combination of antipain (10 μM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze-thawing process.