The objective of this work was to study the in vitro dose-response effect of nitrate (0, 1, 2, 4 and 6 mM) on metabolic hydrogen distribution between rumen fermentation end products. Three 48-h incubations were conducted using bovine rumen contents as an inoculum, and a mixture of hay and concentrate (50:50) as a substrate. Total gas production and composition (methane and hydrogen) were automatically analysed throughout the incubations. Volatile fatty acid and ammonium concentrations were analysed from samples taken after 48 h of incubation. Total gas production was decreased with the highest dose of nitrate (P = 0.002). Methane emissions linearly decreased as the nitrate dose increased (P = 0.005). Kinetics of methane emissions showed that metabolic hydrogen removal via nitrate reduction occurred mainly during the first 10 h of incubation. Gaseous hydrogen production was similar among treatments, despite higher hydrogen emissions for nitrate concentrations >4 mM. Concentrations and proportions of volatile fatty acids were not affected by treatments. The proportion of unaccounted metabolic hydrogen was positive for all treatments, and tended to linearly increase as the nitrate dose increased. In this in vitro work, we confirmed that nitrate is an efficient methane-mitigating compound in the rumen. We also suggest that nitrate or its reduced forms have a direct inhibiting effect towards methanogens, as indicated by the release of gaseous hydrogen and the high efficiency of methane mitigation. However, high nitrate concentrations also decrease overall fermentation.