This work aimed to investigate the effect of energy nature (lipids vs. carbohydrates) on enteric methane emission (eCH4 ) and performance in dairy cows fed grass silage-based diets. Eight multiparous Holstein cows were used in a 4 x 4 Latin square design, with 4 experimental periods of 28 days each. Cows were fed with 4 iso-energetic diets based on grass silage and supplemented with different levels of rapeseed oil (RO) (0, 1.5, 3.0, 4.5% of dietary dry matter (DM), Control, RO-low, RO-medium, RO-high diets, respectively) in substitution of starch from concentrate. Dairy performance, total-tract digestibility, and rumen parameters were measured when animals were in individual respiration chambers for quantifying eCH4. Intake of DM decreased with RO-high compared to other diets. Methane emissions were lowered similarly with all diets containing RO compared to Control (on average, -19% in g/d, -13% in g/kg DMI, -21% in g/kg milk). Ruminal propionate proportion was higher, whereas that of butyrate was lower with RO-high compared to the other diets. Milk yield was higher with RO-low and RO-medium, and was lower for RO-high compared to Control. Milk C16:0 concentration was lower, and C18:1c9, C18:1t11, and other rumen biohydrogenation intermediate concentrations were higher with diets containing RO compared to Control. The shift from the C18:1t11 to C18:1t10 biohydrogenation pathway is in agreement with the milk fat depression observed with RO-medium and RO-high. The inclusion of RO at 1.5% was the best compromise between eCH4, feed efficiency, and milk nutritional quality in cows fed grass silage-based diets. Energy substitution of carbohydrate (mainly starch) by rapeseed oil reduced methane emissions in dairy cows fed grass silage-based diets (up to -23% in g/d) Rapeseed oil content above 3% induced milk fat depression and impaired milk fatty acid profile The inclusion of 1.5% rapeseed oil was the best compromise to reduce methanogenesis (-15% in g/d) and improve dairy performance and milk nutritional quality