The successful cryopreservation of rabbit semen requires the assembly of a diluent with effective permeable and non-permeable cryoprotectants which are correctly quantified. The aim of this study was to evaluate the effects of various dimethyl sulfoxide (DMSO) and sugar concentrations on rabbit spermatozoa during the cryopreservation processes. Four ejaculates from three mature male rabbits (n = 12) were diluted with one of three Tris-based diluents composed of 313.79 mM Tris, 103.07 mM citric acid.H₂O, 50 mM glucose,  80 mg/L kanamcyin and 17% egg yolk plus 3.5%, 7% or 10% DMSO. Diluted samples were cooled to 5°C and frozen in straws using liquid nitrogen and later thawed and kept at 37˚C. Sperm motility and kinetic parameters were assessed using a Computer Assisted Sperm Analyser (CASA), while the integrity of the sperm membrane, acrosome and DNA was calculated using fluorescent staining and flow cytometry. Diluents with lower concentrations of DMSO (3.5%) were shown to have greater cryoprotective effects on motility and kinetic traits (P<0.001), however diluents with 7% and 10% DMSO resulted in significantly higher percentages of sperm with intact membranes (P<0.001). Using 7% DMSO, diluents were then supplemented with either 50 mM glucose, 50 mM sucrose or an amalgamation of both. Diluents with glucose produced sperm with significantly greater post-thaw motility and percentage of intact DNA (P<0.001), however cryopreservation with sucrose produced a significantly higher percentage of sperm with intact acrosomes (P<0.001). Overall, the use of 7% DMSO and low concentrations of sugars in the diluent (50 mM) produced greater post-thaw motility and intact DNA; but diluents with higher sugar concentrations (100 mM) produced significantly higher percentages of sperm with intact acrosomes after cryopreservation. These findings allow for further investigation into the development of an effective media for the cryopreservation and transportation of rabbit semen throughout the farming industry.