In intensively reared dairy cows, milk fat secretion is reduced in response to high-concentrate diets and it is often referred to as the "milk fat depression" (MFD) syndrome. Some trans fatty acid (FA) isomers produced in the rumen of the cows, including t10,c12-18:2, are known for their inhibitory effect on mammary lipogenesis. To study whether this effect depends on the basal diet, duodenal infusions of t10,c12-18:2 were performed on cows fed four different diets (a factorial arrangement of forage:concentrate ratio and linseed oil supplementation). The overall response obtained with t10,c12-18:2 infusion was consistent with previous studies: a decrease in milk fat content and yield without significant variations in milk yield. Mean transfer efficiency of infused t10,c12-18:2 was 19.6%. However, the decrease in milk fat and FA yields (both de novo synthesis and preformed long-chain FA) was less pronounced in cows fed high-concentrate diets (-27% of the initial level), compared with cows fed low-concentrate diets (-42% of initial level). This difference was independent of dietary oil supplementation and milk FA yield before infusion. Results pertaining to effects of dietary forage:concentrate ratio were confirmed by statistical meta-analysis of data from previously published t10,c12-18:2 infusion experiments. This study shows that in cows fed MFD diets the mammary gland becomes more resistant to or experiences a lower response potential to further inhibition of lipogenesis and/or delta-9 desaturation of FA.