The objective of this study was to investigate the profit of L-arginine addition to the freezing extender on freezability, lipid peroxidation, and fertilizing potentials of frozen-thawed buffalo spermatozoa. Semen was collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing different L-arginine concentrations (0, 0.5, 1, 5 and 10 mM) added during the dilution or after equilibration for 2 h. Diluted semen was cooled to 4^oC throughout one hour, equilibrated for 2 h and then frozen in 0.25 ml straws, prior to be stored in liquid nitrogen. Cryopreserved spermatozoa were assessed for post-thawing sperm motility, viability index, acrosomal integrity, lipid peroxidation and fertility rate. The current results clearly indicated that adding 0.5 mM L- arginine to the freezing extender after the equilibration period significantly (P< 0.05) improved post-thawing motility, viability index and maintain acrosomal integrity (61.66±10.14%, 130.83±9.62 and 19.33±4.82%, respectively) compared with the control semen (33.33±4.40%, 88.33±11.03 and 33.67±3.28%, respectively). Moreover, addition of 0.5 mM L- arginine to the freezing extender after equilibration period significantly diminished (P< 0.05) lipid peroxidation (11.33±3.28 nmol/10⁹) compared with the control (26.67±3.18 nmol/10⁹). All of these previously enhanced semen characteristics were reflected positively on its fertilizing potentials. The present results revealed that addition of 0.5 mM L- arginine to the freezing extender after 2 h equilibration (short time exposure) might improve semen quality, preserve the fertilizing potentials and reduce cryodamage of the buffalo spermatozoa.