© CSIRO 2016. This experiment examined which type of oils was a superior suppressor to methane mitigation in ruminants. Four oils, peanut, rapeseed, corn and soybean oils, varying in the contents of unsaturated fatty acids as indicated by their iodine values, were used to investigate their effects on methane production and on the content of the F420 enzyme of ruminal methanogens in an in vitro fermentation. The control group was added with calcium palmitate (100% saturated 16C fatty acid). The results showed that the total gas production over a period of 36 h varied from 20.61 mL to 39.67 mL, and were lower in rapeseed, corn and soybean oil treatments than the control (P <0.05), but not in the peanut oil treatment. The methane concentration in the total gas differed significantly among groups (P <0.05), and decreased with the increases of unsaturation degree of the oils. The coenzyme F420 content, as indicated by F420 fluorescence intensity in supernatant of the medium, was significantly lower in the oil treatments than in the control (P <0.05), and the intensity values decreased with the increases of unsaturation degree of the oils, except for the rapeseed oil treatment. Furthermore, there was a close correlation between F420 content and methane production (r = 0.916). By comparison, soybean oil treatment had higher dehydrogenase activity and bacteria density than the other groups (P <0.05); but was lower in methanogens and genus entodinium (P <0.05), except for the rapeseed oil treatment. Overall, soybean oil contained a high level of unsaturated fatty acids, and could be used as an ingredient of ruminant diets for methane suppression.