The aim of this study was to develop a chemically defined extender for dog sperm cryopreservation by supplementation of essential and non-essential amino acids solution in EY-free PVA extender. Spermatozoa collected from mature dogs (1 ｘ 108 cell/ml) were frozen with EY-free extender supplemented with 0 (control), 1, 2, 4 % essential amino acids (EAAs) or 1, 2, 4 % non-essential amino acids (NEAAs). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing at 37 ℃ for 25 s and post-thaw incubation at room temperature for 20 min. In addition, to evaluate the synergistic effect of EAAs and NEAAs, spermatozoa were frozen with 0, 0.5, 1 or 2 % EAAs-NEAAs mixture (v:v). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing and post-thaw incubation. Additionally, spermatozoa were frozen using EY-free PVA extender supplemented with 2 % EAAs, 2 % NEAAs or 0.5 % EAAs-NEAAs mixture. The ROS level and phosphatidylserine (PS) translocation (Annexin V-FITC assay) were assessed using flow cytometry. In addition, gene expression level for SMCP (motility-related), apoptosis-related BCL2 and BAX was measured after freezing-thawing. The progressive motility of spermatozoa cryopreserved in EAAs or NEAAs significantly increased (P < 0.05) in all groups compared to the control group regardless of thawing conditions. In addition, 1 % NEAAs significantly protected the acrosome membrane of spermatozoa after freezing-thawing (P < 0.05). However, EAAs has shown no significant effect on viability and acrosome membrane integrity of spermatozoa. On the other hand, addition of EAAs-NEAAs mixture to EY-free PVA extender significantly (P < 0.05) increased sperm progressive motility without any effect on viability. Supplementation of 0.5 % EAAs-NEAAs mixture significantly (P < 0.05) increased the expression level of SMCP, BCL2 and BAX compared to control without significant effect on PS translocation and ROS level. We conclude that essential and non-essential amino acids solution can be effectively used in EY-free extender to improve sperm motility, acrosome integrity and gene expression of SMCP and BCL2 in dog sperm cryopreservation.