The aim of this study was conducted to evaluate the effect of sugar kinds and combination of various sugars on acrosome damage of post-thaw spermatozoa in canine. The extender used in this study was Tris-citric acid extender (Tris-buffer) supplemented with 20% Egg-yolk, 8% glycerol, 1% Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer were combined such as control (fructose, xylose, trehalose), two combinations (Fru+Tre, Fru+Xyl, Tre+Xyl) and three combinations (Fru+Tre+Xyl). The acrosome damage rate of post-thaw spermatozoa in Eosin B & Fast Green stain in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fruc+Xyl, Tre+Xyl, Fruc+Tre+Xyl (83.0$\pm$5.6 vs. 82.3$\pm$3.1%, 81.7$\pm$2.1%, 72.0$\pm$2.0%; 80.3$\pm$4.5%, 76.7$\pm$3.8%, 81.0$\pm$5.6). The motility after CASA analysis in Fru+Tre was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose(79$\pm$6 vs 75$\pm$3, 74$\pm$8, 71$\pm$11, 70$\pm$4, 66$\pm$15, 63$\pm$ 12%). However, the progressive motility after CASA analysis in Fru+Tre group was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose (67$\pm$7, 64$\pm$3, 62$\pm$6, 61$\pm$8, 60$\pm$2, 57$\pm$13, 53$\pm$10%). The results indicated that the acrosome damage & progressive motility of post-thaw spermatozoa in 70 mM Fruc+Tre (two combination) following Eosin B & Fast Green stain and CASA analysis.