Objective: Previous studies have shown that mitochondria] DNA (mtDNA) copy number in human sperm is associated with semen quality. This study examines whether there is association between seminal cell free mtDNA copy number and human semen parameters. Study design: Seminal cell-free mtDNA copy number was measured in 205 men. Semen reactive oxygen species (ROS) level was also measured. Sperm quality was assessed using World Health Organization criteria, while sperm motility parameters were determined by computer-aided sperm analysis. Results: Comparing with normozoospermia, patients with asthenozoospermia and oligoasthenozoo-spermia had a significant decrease in cell-free mtDNA copy number and an increase in ROS level in seminal plasma (P < 0.05 for all). Seminal cell-free mtDNA copy number positively correlated with sperm concentration, motility, morphology and motion characteristics (r for 0.247 to 0.673, P < 0.05 for all). Semen ROS level negatively correlated with sperm concentration, motility, and motion characteristics (r for -0.261 to -0.676, P < 0.05 for all). There is a negative relationship between seminal cell-free mtDNA copy number and ROS level (r = -0.573, P < 0.05). Conclusion: The results suggested that seminal cell-free mtDNA copy number is associated with semen parameters, which may serve as a novel diagnostic marker of semen quality. (C) 2018 Elsevier B.V. All rights reserved.