Cryopreservation procedures negatively affect the quality traits of sperm, causing certain changes at structural and molecular levels due to thermal, mechanical, osmotic, and oxidative damage. The objective of this study was to examine the potential of canine adipose-derived mesenchymal stem cells (Ad-MSCs) for providing protection to the dog sperm against cryo-damage. Canine Ad-MSCs were selected on the basis of the significantly higher gene expression for different proteins actively involved in the cell repair including annexin 1 (ANX1), histone H3 (H3) and high mobility group B (HMGB) protein compared to skin fibroblasts. Semen was collected from four healthy dogs by digital manipulation. The washed pooled ejaculates were diluted with buffer 2 (extender) supplemented without Ad-MSCs (Control), with 2.5 × 106 Ad-MSCs/mL (Group 1) or with 5 × 106 Ad-MSCs/mL (Group 2). Group 1 exhibited significantly higher post-thaw motility, live sperm, intact plasma membrane and normal acrosomes than the other groups. Additionally, Group 1 showed significantly higher expression levels of genes related to the repair of membranes (ANX1, dysferlin; DYSF, and fibronectin; FN1) and chromatin material (H3 and HMGB). Protein expression of ANX1, H 3, and FN1 was also statistically more in Group 1 than in Control. The results confirm that canine Ad-MSCs can effectively preserve the quality of frozen-thawed sperm by a reduction in cryoinjury. At an appropriate concentration, Ad-MSCs significantly improve the quality of post-thaw dog sperm.