Previous studies in our laboratory have shown a positive correlation between oxidative stress and stallion fertility; leading to the hypothesis that oxidative phosphorylation (OXPHOS) provides the majority of energy for stallion sperm motility and produce ROS as a by-product. This contrasts with spermatozoa of many other species, including human, which rely predominantly on glycolysis. The aim of this study was to investigate the effects OXPHOS inhibition on the motility of horse spermatozoa using the electron transport chain inhibitor diphenyleneiodonium (DPI). To ensure that glycolysis was uninhibited, the effect of DPI on human sperm motility was also assessed. Horse (n=3) and human (n=3) spermatozoa were diluted in BWW (20 × 106/mL) and incubated at 37°C ± DPI (10µM). Motility (CASA) of horse and human spermatozoa, and vitality and ATP production of horse spermatozoa were measured after 1 h. Treatment of horse spermatozoa with DPI caused a decline in velocity and %Rapid cells (VAP: 141.3 to 38.4 µm/s; VSL: 109.3 to 27.1 µm/s; VCL: 247.2 to 97.6 µm/s; %Rapid: 65.3 to 2.7%), and significantly decreased ATP production (0.035 to 0.008 µg/2×106 spermatozoa), while vitality remained unaffected. As there was no decrease in velocity or %Rapid cells following DPI treatment of human spermatozoa, it was concluded that glycolysis was unaffected. Velocity parameters of untreated horse spermatozoa were all higher (p≤0.05) than those of human spermatozoa (VAP: 141.3 vs. 87.8 µm/s; VSL: 109.3 vs. 76.0 µm/s; VCL: 247.2 vs. 146.4 µm/s respectively). This is the first report indicating that stallion spermatozoa are predominantly dependent on OXPHOS for ATP production. This metabolic pathway is likely to be responsible for the high rates of oxidative stress in horse spermatozoa, the faster velocity of horse compared to human spermatozoa, and the positive correlations between oxidative damage and fertility which are observed in horses, but not humans.