Background: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H₂) + carbon dioxide and from H₂ + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H₂ that accumulates less than expected under inhibited methanogenesis. Results: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H₂ concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H₂-producing bacteria to regulate the amount of H₂ production. No previously reported alternative H₂ sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. Conclusion: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H₂ metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen.