Canine seminal plasma contains antioxidant enzymes to protect sperm against internally generated ROS. These enzymes are removed from seminal plasma during the process of cryopreservation. The freezing/thawing process can cause some morphological and functional changes via ice crystallization and osmolality imbalance. The present study was conducted to evaluate the effects of curcumin supplementation on sperm total count, motility, progressive motility, viability, morphology, total antioxidant capacity (TAC), DNA integrity and NOX5 gene expression of dog frozen semen. The pooled semen was allocated to fresh (Group 1) and frozen (Group 2) controls, curcumin (2.50 mM) (Group 3) and curcumin (5.00 mM), (Group 4). Sperm parameters including total sperm count, morphology, motility, progressive motility, sperm concentration and DNA integrity in addition to TAC were evaluated in fresh and frozen-thawed semen samples. Real-time RT-PCR was used to investigate NOX5 and GADPH (reference gene) genes expressions. Curcumin at 2.50 mM provided a greater protective effect on the DNA integrity compared to 5.00 mM and control groups. TAC was significantly higher in 2.50 mM group than other groups. NOX5 gene expression in curcumin 2.50 mM was higher than 5.00 mM group. In conclusion, curcumin seems to emolliate sperm parameters and to protect sperm against sperm reactive oxygen stress and increases NOX5 gene expression.