Nanoparticles of cerium oxide (nano-ceria) possess reactive oxygen species(ROS) scavenging properties as they mimic superoxide dismutase and catalase in biological systems. Therefore, present study aimed to investigate the effects of incorporating nano-ceria in the cryo- diluent on post-thaw sperm antioxidant profile and quality attributes in water buffalo. Semen samples from five maturebulls were collected and evaluated. Qualified samples were pooled, divided into5 aliquots and diluted in Tris-citric acid extender containing differential concentrations of nanoparticles (0.14 µmol L-1, 0.29 µmol L-1, 0.43 µmol L-1,0.58 µmol L-1 and negative control). Samples were cooled to 4 oC for 120 minand equilibrated at 4 oC for 4 hours, packaged in 0.54 mL French straws, frozen from initial holding at +4°C for 2 min, from +4°C to -20°C at 10 ºCmin-1, from -20°C to -100°C at 30°C min-1 and final holding for 1 min at-100°C with programmable freezer and stored in liquid nitrogen at -196 oC. At post-thaw, various in vitro sperm antioxidant enzyme profiles and other quality parameters were determined. At post-thawing, levels of catalase (0.28 ± 0.015unit mg-1) peroxidase (0.24 ± 0.003 unit mg-1) and reduced glutathione (40.40± 2.12 µmol L-1) were highest (P< 0.05) with 0.43 µmol L-1 of nano-ceria as compared to negative control and other treatment groups. Moreover, lipid peroxidation (0.79 ± 0.02 nmol L-1 min-1 mg protein- 1) level was lowest (P<0.05) with 0.43 µmol L-1 of nano-ceria. Results also revealed that 0.43 µmol L-1exhibited higher (P < 0.05) sperm progressive motility (31.51 ± 1.51), rapid velocity (40.12 ± 1.90), average path velocity (78.00 ± 1.20), straight linear velocity (65.23 ± 1.28), curved linear velocity (101.2 ±3.2), supra-vital plasma membrane integrity (62.31 ± 1.28), viability (56.82 ± 2.01) and DNA integrity(97.00 ± 2.05) than control and other treatment groups. We concluded that the0.43 µ mol L-1 of Nano-ceria in cry diluent improved the post-thaw quality of water buffalo spermatozoa in terms of elevated levels of endogenous antioxidant enzymes, improved structural and functional integrity.