The aim of this study was to investigate the effect of adding homologous or heterologous bovine seminal plasma (SP) to SP-free sperm samples before freezing on sperm quality after thawing. Ejaculates from bulls of known fertility were used as a source of SP. The SP was removed from further aliquots of the same ejaculates by colloid centrifugation to create SP-free sperm samples; the resuspended sperm pellets were treated with homologous or heterologous SP from high or low fertility bulls at 0%, 1% or 5% before freezing. After thawing, sperm quality was evaluated by computer-assisted sperm analysis and flow cytometry for membrane integrity, reactive oxygen species, chromatin structure, mitochondrial membrane potential and protein tyrosine phosphorylation. Data were analysed using Proc MIXED, SAS®. Post-hoc comparisons were adjusted for multiplicity using Tukey’s method. The addition of SP resulted in significant differences in sperm quality, namely velocity class A, Velocity Straight Line (VSL), Velocity Average Path (VAP), Velocity Curved Line (VCL), Amplitude of Lateral Head Displacement (ALH), Hyperactive (HYP), reactive oxygen species (ROS) production and % DNA fragmentation index (DFI) (P<0.05 for each). Although adding 5% homologous SP from high fertility bulls was beneficial to sperm kinematics, 5% heterologous SP from high fertility bulls had a deleterious effect on chromatin integrity and on sperm velocity. In conclusion, adding SP may have either a beneficial effect or a deleterious effect depending on the individuals involved. It might be feasible to use this method to improve sperm quality in some circumstances.