The aim of present the study was to improve post-thawing canine sperm quality using OptiprepTM (60% iodixanol in water). The pooled semen was centrifuged and sperm pellet was resuspended with the first buffer (Tris [hydroxymethyl] amino methane, citric acid, fructose, kanamycin sulfate in distilled water). The sperm suspension was added by equal volume of second buffer (egg yolk, glycerol and first buffer) which was made with 0% and 2.5% OptiprepTM. After 1 week, frozen-thawed sperm of each groups were assessed by Chromomycin staining (CMA3), quantitative real time PCR (RT-qPCR), and mucus penetration test. For statistical analysis, t-test was performed using GraphPad Prism 5.0 and all differences were considered significant at P < 0.05. As the results, the protamine deficiency in 2.5% sperm treatment group significantly lower (54.0 ± 0.9%) than control (46.6 ± 2.2%). In RT-qPCR result, the sperm treated with 2.5% OptiprepTM showed significantly higher expression of protamine gene (PRM2 and PRM3) and sperm acrosome associated-3 gene (SPACA3) than control. The mucus penetration test result of post-thawing sperm showed that sperm in 2.5% OptiprepTM treated group (56.4 ± 5.3%) was significantly higher than control (40.7 ± 4.7%). In conclusion, the 2.5% OptiprepTM could protect and improve post-thawing sperm quality. For the further studies, adipose-derived stem cells will be used to improve the fertilization ability of canine frozen-thawed spermatozoa. This study was supported by Cooperative Research Program of RDA (CCAR, #PJ013954022018) Research Institute for Veterinary Science, and the BK21 plus program.