OBJECTIVE The study is to develop a glycerol-free extender using glucose for canine sperm cryopreservation,. METHODS Tris [hydroxymethyl] aminomethane (TRIS) extender. Canine sperm were cooled to 4 degree C in TRIS containing 44.4 mM glucose for 100 min, and then cooled at 4 degree C in TRIS with glucose concentrations of 0 mM, 44.4 mM, 100 mM, 200 mM, or 300 mM for 30 min followed by cryopreservation. After thawing at 37 degree C for 25 sec, sperm motility, viability, and morphological abnormalities were evaluated. In addition, 300 mM glucose-TRIS was compared to TRIS extenders with a final concentration of 5 % glycerol. Sperm phosphatidylserine (PS) translocation after freezing and thawing was assayed by flow cytometry using Annexin V-FITC apoptosis detection kit. RESULTS Progressive motility and viability (42% and 41%, respectively) were significantly higher in the 300mM group than the other groups with lower concentrations of glucose (P < 0.05). PS translocation index was significantly lower in 300mM glucose-TRIS than those in extenders with glycerol (85 vs 93, P < 0.05). CONCLUSIONS These results indicate that cryopreservation of canine sperm using glycerol-free 300 mM glucose-TRIS is feasible and yields more motile sperm with lower PS translocation compared with extenders containing glycerol.