To our knowledge, the roles of kaempferol in semen extension or cryobiology haven't been discovered until now. Furthermore, the intracellular signals triggered by kaempferol in buffalo spermatozoa during semen processing haven't been reported. So, the current study intended to clarify the role of kaempferol during the cryopreservation of buffalo spermatozoa. Six buffalo bulls were adopted for semen collection. Two consecutive ejaculates were collected from each buffalo bull weekly using an artificial vagina. The ejaculates were pooled to eliminate variability. Semen extension was done using the Tris-based diluent supplemented with Kaempferol at concentrations of 5, 10, 15, 25, 50, and 100 µg/ml vs. tris-based diluent only as control. Samples were subjected to freezing then thawing to evaluate the vital sperm characteristics. It was discovered that kaempferol at a concentration of 25 and 50 µg/ml of significantly (p<0.05) improved the post thawing motility, acrosomal integrity, viability index, glutathione reductase activity, superoxide dismutase and total antioxidant capacity. Moreover, it significantly (p<0.05) decreased the rate of lipid peroxidation, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and DNA fragmentation (%). In conclusion, kaempferol supplementation to buffalo semen extender at 25 and 50 µg/ml significantly enhanced its quality by reducing the cryo-damage rate depending on their enzymatic and non-enzymatic potentials.