The endocannabinoid system (ECS) participates in regulating whole body energy balance. Overactivation of the ECS has been associated with the negative consequence of obesity and type 2 diabetes. Since activators of the ECS rely on lipid-derived ligands, an investigation was conducted to determine whether dietary PUFA could influence the ECS to affect glucose clearance by measuring metabolites of macronutrient metabolism. C57/blk6 mice were fed a control or DHA-enriched semi-purified diet for a period of 112 d. Plasma, skeletal muscle, and liver were collected after 56 d and 112 d of feeding the diets for metabolomics analysis. Key findings characterized a shift in glucose metabolism and greater catabolism of fatty acids in mice fed the DHA diet. Glucose use and promotion of fatty acids as substrate were found based on levels of metabolic pathway intermediates and altered metabolic changes related to pathway flux with DHA feeding. Greater levels of DHA-derived glycerol lipids were found subsequently leading to the decrease of arachidonate-derived endocannabinoids (eCB). Levels of 1- and 2-arachidonylglcerol eCB in muscle and liver were lower in the DHA diet group compared to controls. These findings demonstrate that DHA feeding in mice alters macronutrient metabolism and may restore ECS tone by lowering arachidonic acid derived eCB.