Modifying milk fat composition to enhance its content of valuable fatty acids (FA) is required to meet the needs of a society which is becoming better informed about the relationship between diet and health. Manipulating the cow’s diet is an effective, natural way to modify the amount and composition of milk fat of cows. The two main factors that affect the cow’s diet concern management system (MS; pasture vs. confinement), and supplementation of diets with lipid supplements. Marine oils specifically are fed to enhance milk with n-3 long-chain polyunsaturated FA (n-3 LC-PUFA). The effects of source of marine lipid supplement (LS; fish oil vs. microalgae) in the cow’s diet and its interaction with MS on milk fat composition have not been studied. Thus, the main objective was to determine the interaction of MS and LS on milk FA profile and on expression of lipogenic genes in mammary, adipose and liver of lactating dairy cows. Compared with cows in confinement, grazing cows produced milk fat with lower content of unfavorable FA (12:0-16:0), while increasing the levels of beneficial FA including cis-9 18:1, 18:3 n-3 and conjugated 18:2. Feeding either fish oil or microalgae improved levels of n-3 LC-PUFA and reduced those of 16:0 in milk fat regardless of MS, but concurrently increased the level of other trans 18:1 isomers at the expense of trans-11 18:1. The reduced secretion of 12:0-16:0 in milk from grazing compared with confined cows was associated with lower mammary expression of lipogenic genes suggesting that part of the effect of MS on milk FA profile is mediated transcriptionally. The effect of LS on lipogenic gene expression was tissue specific with the greatest response to treatment observed in liver despite its minor role in lipogenesis in cattle relative to the mammary and adipose. Major conclusions were that milk produced in pasture systems has a more healthful FA profile than that of confinement systems, and that MS and LS have tissue specific effects on lipogenic gene expression in dairy cattle which have important effects on cow performance and healthfulness of the milk FA profile.