Cryopreservation induces sub lethal damage to the spermatozoa, thereby reduce their fertile life. There are some biochemical additives that may enhance buffalo semen freezability; L-carnitine is one of these biochemical semen additives. Till now, the exact effects of L-carnitine on buffalo semen processing outcomes haven't been discovered. The current study aimed to clarify L-carnitine roles during buffalo semen cryopreservation. Semen was cryopreserved in tris-based extender supplemented with different concentrations of L- carnitine (0.01, 0.05 and 0.1mg/ml)Vs.Tris-based extender only (control). Then they were processed to cryopreservation and thawing to assess different semen characteristics. Cryopreserved semen was assessed for percentage post- thawing motility, acrosomal and plasma membrane integrity, viability, DNA damage, antioxidant enzymes concentration, lipid peroxidation, in vitro fertilizing potentials and conception rate. Current results indicated that addition of 0.05 mg/ml L- carnitine to semen extender significantly (P<0.05) improved post-thawing motility, viability and acrosomal integrity (63.33±9.28 %, 133.33±9.40 and 12.33± 2.02 %, respectively) compared with control (43.33±6.01 %, 74.16± 10.93 and 24.67±2.03 %, respectively). Moreover, 0.05mg/ml L- carnitine significantly increased (P<0.05) total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) concentrations (0.50±0.07 mµ/ml, 73.67±5.37 U/ml and 106.66±12.03 U/L, respectively) with respect to the control (0.19±0.01 mµ/ml, 27.33±3.49 U/ml and52.33±4.09 U/L, respectively). Furthermore, 0.05mg/ml L- carnitine significantly decreased (P<0.05) lipid peroxidation of the cryopreserved spermatozoa compared with the control semen (11.00±1.73 vs 24.82±4.90nmol/ml). Likewise, at this concentration sperm DNA damage, tail length and tail moment of the cryopreserved semen significantly (P<0.05) reduced (1.87±0.36 %, 1.83±0.33 µm and 3.72±1.44, respectively) compared with control (3.47±0.13 %, 3.48±0.17 µm and 11.91±0.87, respectively). Additionally, 0.05 mg/ml L- carnitine significantly (P<0.05) improved in vitro fertilization rate (57.45%) and conception rate (61.76%) compared with the control (33.33 and 37.93%, respectively). In conclusion the use of 0.05 mg/ml L-carnitine in the freezing extender improves DNA integrity through enhancing the antioxidant defense of buffalo sperm cells and decreasing the rate of lipid peroxidation. Therefore, L-carnitine may improve sperm cryopreservation quality, reduce cryodamage and improve sperm fertilizing potential