Background: The metabolite composition of cow milk is dependent on a large variety of animal associated factors including diet, genotype and gut microbiome composition. The objective of this study was to investigate changes in cow milk polar metabolite composition resulting from dietary and DGAT1 (Diacylglycerol O-acyltransferase 1) genotype perturbations. Methods and Results: Cows were fed a standard diet and a diet supplemented with (poly)unsaturated fatty acids (experimental diet) for ten weeks. Metabolite profiles were determined using 1H NMR (1-Hydrogen Nuclear magnetic resonance) technology. The results showed that the diet affected the polar metabolite composition of milk via the metabolism of the cow and via the metabolism of the gut and rumen microbiota. The experimental diet reduced the metabolic rate, especially the energy metabolism and the amino-sugar and amino acid metabolism, of the cows. Conclusion: Our results suggests the DGAT1 genotype affects both the diet related polar metabolite metabolism of the cow as well as that of the rumen microbiota. Milk metabolite levels in animals with more DGAT1 A-alleles were higher than milk metabolite levels in animals with more K-alleles.