This study was conducted to evaluate the effect of sugar kinds and combination of various sugars, and straw size and pre-freezing time on motility of post-thaw spermatozoa in canine. In general, the extender was supplemented with fructose or glucose for semen freezing. 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+Xy1, Tre+Xy1) and three combinations (Fru+Tre+Xy1). The motility after CASA analysis in Fru+Tre+Xy1 was higher than those in fructose, trehalose, xylose, Fru+Tre, Fru+Xy1, Tre+Xy1 (69 vs. 58, 61, 50, 65, 20, 54). However, the progressive motility after CASA analysis in Fru+Tre group was higher than those in fructose, trehalose, xylose, Fru+Xy1, Tre+Xy1, Fru+Tre+Xy1 (59% vs. 47, 55, 42, 13, 49, 44%). The survival rate of post-thaw spermatozoa in 0.25 $m\ell$ straw for 10 min pre-freezing was significantly higher than those in 0.25 $m\ell$ straw for 10 min, 0.25 and 0.5 $m\ell$ for 5 min (80+0.0 vs. 65+7, 68+16, 58+8%; P<0.05). The results indicated that the progressive motility of post-thaw spermatozoa in 70 mM Fru +Tre (two combination) following CASA analysis and 0.25 ml straw for 10 min pre-freezing time could be better for freezing of semen in canine.