In this study the influence of modulated concentrate-to-roughage ratio on the fatty acid profile of milk fat was investigated in dairy cows. Therefore, corn stover was compared with better-quality roughages. Two groups of in total 24 Holstein dairy cows (136 ± 37 days in milk) received either a high-forage diet (Diet MF, forage-to-concentrate ratio [F:C] = 60:40) with alfalfa hay, corn silage and Chinese wild rye as forage sources, or a low-forage diet with corn stover as forage source (Diet CS, F: C = 40:60) for an experimental period of nine weeks. During the study, milk yield as well content and fatty acid profiles of milk fat were examined. Dietary treatments had no effect on milk yield and milk fat content, whereas dry matter intake (p < 0.01) and milk fat yield (p < 0.05) were higher for Diet MF than for Diet CS. Compared with Diet CS, feeding Diet MF increased the daily intake of total unsaturated fatty acids and the C18:0 and C18:3 contents (p < 0.01) in milk fat, whereas the total content of fatty acids <16C was decreased (p < 0.05). No influence on total saturated, monounsaturated and polyunsaturated fatty acids in milk was observed. The ratio of total unsaturated fatty acids in milk fat to its daily intake was substantially lower for Diet MF compared with Diet CS, suggesting that the high proportion of roughage resulted in a high rate of biohydrogenation in the rumen.