Recently, male impact has been recognized as a momentous cause of infertility. Male infertility isn't an entity but it evokes a variety of mechanisms and explanations to understand it. In between, a biochemical semen additive that improves or overcome certain semen situations so can enhance or treat semen fertility and infertility, respectively. 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 samples were obtained from six fertile buffalo bulls (aged 3 to 5 years). Weekly, two consecutive ejaculates were collected from each bull for successive six weeks using artificial vagina. The ejaculates were pooled to eliminate samples variability. Semen samples were extended with Tris-based extender supplemented with different concentrations of L-carnitine (0.01, 0.05 and 0.1 mg/ml) Vs. Tris-based extender only (control). Then they were processed to cryopreservation and thawing to assess different semen characteristics. It had been found that L-carnitine (0.05mg/ml) significantly (p < 0.05) improved post thawing motility, viability index, acrosomal integrity, in vitro fertilization, blastocyst and conception rates of treated buffalo semen (57.00±2.55%, 126.5±8.33, 14.40 ± 1.43%, 56.45%, 15.79% and 64.71%, respectively), compared with control (42.00±2.56%, 87.00±10.43, 23.80±1.86%, 36.53%, 2.13% and 42.86%, respectively). Conclusion: L-carnitine supplementation to buffalo semen extender significantly enhanced its characteristics and protecting its plasma membrane and mitochondrial functional integrity. Moreover, the preceding results focusing more light on the potential roles of L-carnitine in regulating male fertility and infertility.