The present study has examined the effect of different concentrations (1 μg/ml, 10 μg/ml and 100 μg/ml) of titanium oxide (TiO₂) nanoparticles (NPs) (<100 nm) on viability, membrane integrity, capacitation status and DNA integrity of buffalo spermatozoa. Characterization of NPs was done by the transmission electron microscopy (TEM) and dynamic light scattering (DLS). Sperm chromatin dispersion (SCD) test and acridine orange test (AOT) were employed to detect DNA fragmentation in sperm treated with NPs. There was significant (p < 0.05) decrease in cell viability and membrane integrity (assessed by enzyme leakage) at 6 h of incubation with NPs. However, significant (p < 0.05) increase in sperm capacitation was observed for TiO₂ NP albeit at lower concentrations. In DNA fragmentation assay, there was dose-dependent increase in the DNA fragmentation (r = 0.96). Ultrathin cross-sections revealed TiO₂ NPs inside head and plasma membrane of the buffalo spermatozoa as assessed by TEM. These studies suggest that TiO₂ NPs may have cytotoxic effect on buffalo spermatozoa by affecting sperm functionality and causing high amount of DNA fragmentations.