Optimizing milk production efficiency implies diets allowing low methane (CH4) emissions and high dairy performance. We hypothesize that nature of energy (starch v. lipids) and lipid supplement types (monounsaturated fatty acid (MUFA) v. polyunsaturated fatty acid (PUFA) mitigate CH4 emissions and can induce low milk fat content via different pathways. The main objective of this experiment was to study the effects of starch-rich or lipid-supplemented diets that induce milk fat depression (MFD) on rumen biohydrogenation (RBH) of unsaturated fatty acids (FA) and enteric CH4 emissions in dairy cows. Four multiparous lactating Holstein cows (days in milk=61±11 days) were used in a 4×4 Latin square design with four periods of 28 days. Four dietary treatments, three of which are likely to induce MFD, were based (dry matter basis) on 56% maize silage, 4% hay and 40% concentrates rich in: (1) saturated fatty acid (SFA) from Ca salts of palm oil (PALM); (2) starch from maize grain and wheat (MFD-Starch); (3) MUFA (cis-9 C18:1) from extruded rapeseeds (MFD-RS); and (4) PUFA (C18:2n-6) from extruded sunflower seeds (MFD-SF). Intake and milk production were measured daily. Milk composition and FA profile, CH4 emissions and total-tract digestibility were measured simultaneously when animals were in open-circuit respiration chambers. Fermentation parameters were analysed from rumen fluid samples taken before feeding. Dry matter intake, milk production, fat and protein contents, and CH4 emissions were similar among the four diets. We observed a higher milk SFA concentration with PALM and MFD-Starch, and lower milk MUFA and trans-10 C18:1 concentrations in comparison to MFD-RS and MFD-SF diets, while trans-11 C18:1 remained unchanged among diets. Milk total trans FA concentration was greater for MFD-SF than for PALM and MFD-Starch, with the value for MFD-RS being intermediate. Milk C18:3n-3 content was higher for MFD-RS than MFD-SF. The MFD seems more severe with MFD-SF and MFD-RS than PALM and MFD-Starch diets, because of a decrease in milk SFA concentration and a stronger shift from trans-11 C18:1 to trans-10 C18:1 in milk. The MFD-SF diet increased milk trans FA (+60%), trans-10 C18:1 (+31%), trans-10,cis-12 CLA (+27%) and PUFA (+36%) concentrations more than MFD-RS, which explains the numerically lowest milk fat yield and indicates that RBH pathways of PUFA differ between these two diets. Maize silage-based diets rich in starch or different unsaturated FA induced MFD with changes in milk FA profiles, but did not modify CH4 emissions.