Cryopreservation induces sublethal damage to the spermatozoa, which leads to reduce their fertile life. The objective of the present study was to investigate the effect of trehalose, cysteine and hypotaurine on freezability, ultrastructure and in vitro fertilizing potentials of the buffalo spermatozoa. Buffalo spermatozoa were cryopresreved with Tris egg yolk extender containing 7% glycerol suplemmented with100 mM trehalose, 100 mM trehalose + 5 mM cysteine, 100 mM trehalose + 25 mM hypotaurine, 100 mM trehalose + 5 mM cysteine + 25 mM hypotaurine or Tris-based extender only (control).  Cryopresreved spermatozoa were assessed for post-thawing sperm motility; viability and acrosomal integrity, ultrastructure changes, biochemical activity and in vitro fertilizing potentials. The current results clearly indicated that adding a mixture of 100 mM trehalose , 5 mM cysteine and 25 mM hypotaurine to Tris extender significantly improved (P<0.05) post-thawing sperm motility, viability index and maintained acrosomal integrity following cryopreservation (63.33±7.27%, 155.83±21.06and 12.33±2.73%, respectively) compared with the control spermatozoa (38.33±4.41%, 94.17±12.28 and 25.33±3.49%, respectively). The current results illustrated that addition of combination of additives protected the plasma membrane, acrosomal region and mitochondria and maintained the ultrastructure integrity of the cryopresreved spermatozoa compared with the control spermatozoa. Additionally, the current results revealed that combination of trehalose, cysteine and hypotaurine significantly increased the total antioxidant capacity level (TAC), superoxide dismutase activity (SOD) and glutathione reductase activity (GSH) and reduced  significantly the lipid peroxidation rate (0.60±0.89 mµ/ml, 70.00 ±5.78 U/L, 81.66±4.41 U/L and 7.33±1.86 nmol/ml, respectively) compared with the control extender (0.23±0.04 mµ/ml, 34.67±6.74U/L, 51.67±14.82 U/Land 20.67±3.84 nmol/ml, respectively). Furthermore, a mixture of trehalose, cysteine, hypotaurine significantly improved (P<0.05) the rate of in vitro fertilization, cleavage and embryo development to morula and blastocyst stages (62.66±6.28, 53.54±3.89, 23.87±6.28 and17.94±2.57%) compared with the control extender (31.19±4.42, 18.47±5.04, 8.36±1.91 and .42±1.71%, respectively). Therefore, the present results revealed that addition of a mixture of trehalose, cysteine and hypotaurine to the freezing extender could improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.