There is scarce information on whether inhibition of rumen methanogenesis induces metabolic changes on the host ruminant. Understanding these possible changes is important for the acceptance of methane-reducing practices by producers. In this study we explored the changes in plasma profiles associated with the reduction of methane emissions. Plasma samples were collected from lactating primiparous Holstein cows fed the same diet with (Treated, n = 12) or without (Control, n = 13) an anti-methanogenic feed additive for six weeks. Daily methane emissions (CH4, g/d) were reduced by 23% in the Treated group with no changes in milk production, feed intake, body weight, and biochemical indicators of health status. Plasma metabolome analyses were performed using untargeted [nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS)] and targeted (LC-MS/MS) approaches. We identified 48 discriminant metabolites. Some metabolites mainly of microbial origin such as dimethylsulfone, formic acid and metabolites containing methylated groups like stachydrine, can be related to rumen methanogenesis and can potentially be used as markers. The other discriminant metabolites are produced by the host or have a mixed microbial-host origin. These metabolites, which increased in treated cows, belong to general pathways of amino acids and energy metabolism suggesting a systemic non-negative effect on the animal.