The rumen simulation technique (RUSITEC) was used to evaluate the effect of two levels of echium (Echium plantagineum) oil (EO) on methano-genesis. Treatments comprised: control (commercial total mixed ration for lactating dairy cows; positive control), control supplemented with sodium 2-bromoethanesulfonate (5 mM on a dry matter (DM) basis; negative control; BES) and control supplemented with 4% (EO4) and 8% (EO8) of echium oil (g · 100^–1 g DM). Methane production and the proportion of methanogens in total rumen prokaryote 16S rDNA from effluents and solid residues were decreased by EO8. Oil supplementation did not affect total volatile fatty acid production and digestibility of DM and neutral detergent fibre, but increased propionate proportion. Despite the in vitro results suggesting that echium oil is a promising natural strategy for the mitigation of enteric methane, it is difficult to distinguish whether the observed effects are due to secondary metabolites, a particular fatty acid, or mixtures of fatty acids contained in the echium oil