The effect of linseed oil, DGAT1 K232A polymorphism, and the interaction between linseed oil and DGAT1 K232A polymorphism on methane (CH4) emission, and on energy and nitrogen (N) metabolism, lactation performance, ruminal fermentation, and rumen microbial composition was investigated. The experiment involved 24 lactating Holstein-Friesian cows (i.e. 12 with DGAT1 KK genotype and 12 with DGAT1 AA genotype), and followed a cross-over design with two dietary treatments: control diet (CON), and linseed oil diet (LSO) with a difference of 22 g/kg dry matter (DM) in fat content between the two diets. Both diets consisted of 40% corn silage, 30% grass silage, and 30% concentrates, on DM basis. Apparent digestibility, lactation performance, N and energy balance, and CH4 emission were measured in climate respiration chambers. The DGAT1 K allele was associated with a lower milk yield, lactose yield, and proportion of poly-unsaturated fatty acids (FA) in milk fat, and a higher milk fat and protein content, and proportion of saturated FA in milk fat compared with the DGAT1 A allele. DGAT1 K232A polymorphism did not affect CH4 or H2 emission, ruminal fermentation or ruminal microbial concentrations. Rumen microbial composition was also unaffected in terms of the whole microbial population, however at the genus level the relative abundances of certain bacterial genera were found to be affected by the DGAT1 K232A polymorphism. The DGAT1 K allele was associated with a lower crude fat digestibility and metabolizability (i.e. ratio of metabolizable to gross energy intake), and with a tendency for a lower gross energy and NDF digestibility, and milk N efficiency compared with the DGAT1 A allele. Diet LSO decreased CH4 production (g/d) by 8%, CH4 yield (g/kg DM intake) by 6%, and CH4 intensity (g/kg fat- and protein-corrected milk) by 11%, but did not affect H2 emission. Diet LSO also decreased ruminal acetate molar proportion, the acetate to propionate ratio, and the archaea to bacteria ratio, whereas ruminal propionate molar proportion and milk N efficiency increased. Ruminal microbial composition tended to be affected by diet in terms of the whole microbial population, with certain bacterial genera found to be significantly affected by diet. These results indicate that the DGAT1 K232A polymorphism does not affect enteric CH4 production and production pathways, but that it does affect traits other than lactation characteristics, including nutrient digestibility, metabolizability, and the relative abundance of certain rumen bacterial genera. Additionally, linseed oil reduces CH4 emission independent of DGAT1 K232A polymorphism and affects the rumen microbiota and its fermentative activity.