The purpose of this study was to investigate the effect of different doses of trehalose and cysteine on bull frozen-thawed sperm quality. In this study, semen samples were collected from three healthy mature Holstein bulls by an artificial vagina. After primary evaluations, the samples were pooled to eliminate individual effects. Then, semen samples were divided into six equal parts and each part was extended and frozen with one of the following extenders. 1) without cysteine and trehalose (T0C0), 2) Five mM cysteine without trehalose (T0C5), 3) Ten mM cysteine without trehalose (T0C10), 4) A hundred mM trehalose without cysteine (T100C0), 5) Five mM cysteine with 100 mM trehalose (T100C5), 6) Ten mM cysteine with 100 mM trehalose (T100C10). Motility parameters, acrosome and membrane integrity and phosphatidylserine translocation assay were evaluated after thawing. All data were analyzed using a 2×3 factorial trail following data collection. The results of this study showed that cysteine had no effect on motion characteristics, apoptotic status, acrosome integrity and viability (P≥ 0. 05). Also motion parameters, viability, acrosome and membrane integrity, in group without trehalose was higher than group containing that (P≤ 0. 05). Regarding to interactive effects, the T0C10 group had higher amount of viability (P≤ 0. 05) and lower necrotic sperms compared to rest of the groups. Based on the results, it can be concluded that cysteine had no remarkable effect and addition of Trehalose could negatively affects post-thawed bull sperm quality.