Nitrate may reduce the ruminal methane emission by competing methanogenesis to achieve more hydrogen. For this purpose, twenty Holstein lactating cows were examined using a 2×2 factorial design in 4 groups for 60 days with two forage levels (40% and 60%) and supplemental nitrate 0% (F40 and F60) and 3.5% (F40N and F60N) of diet dry matter (DM). Then, the effect of nitrate and forage levels on cow performance, ruminal fermentation, methane emission, and metabolic hydrogen sink were evaluated. The nitrate supplementation did not significantly affect milk yield and ECM/DMI, while milk urea nitrogen was increased. Lowest quantity of milk vitamins (A and E) was observed in nitrate groups. The nitrate supplementation increased c9-C18:1, unsaturated fatty acids, and n-6/n-3 contents of the milk. Blood parameters were affected by nitrate supplementation. Blood met-Hb concentration was increased, while blood glucose was decreased in nitrate groups. High forage and nitrate fed animals (F60N) had higher ruminal acetate and lower propionate concentration, and higher acetate+butyrate to propionate ratio than other groups. Nitrite and NH3-N concentrations were higher in the rumen of nitrate fed animals. Nitrate supplementation inhibited gas volume and methane emission without affecting volatile fatty acids at 12 and 24 h of incubation. The H2 balance, H2 production and consumption, and recovery percentage were significantly lower in F60N group. In conclusion, nitrate supplementation can be employed as an alternative strategy for improving ruminal fermentation, milk quality and methane inhibition.