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Background: Dietary supplementation of omega-3 fatty acids can reduce the activation of the endocannabinoid system（ECS） by decreasing the availability of arachidonic acid, thus lowering endocannabinoids（e CBs） levels. The ECS is a modulator of energy metabolism, stress response and inflammation in mammals, yet there is little information on the roles of the ECS in transition dairy cows. During the periparturient period, the adipose tissue and liver are the main metabolic organs that participate in the adaptations of dairy cows to onset of lactation; however, exceeded adipose tissue lipolysis and accumulation of lipids in the liver have adverse effects on cows’ physiology. Here we aimed to examine whether omega-3 supplementation during the transition period will modulate ECS activation and affect metabolic and inflammatory indices in postpartum dairy cows, by supplementing twenty-eight transition Holstein dairy cows with either saturated fat（CTL） or encapsulated flaxseed oil（FLX）. Components of the ECS, metabolic and inflammatory markers were measured in blood, liver, and subcutaneous adipose tissue.Results: FLX supplementation reduced feed intake by 8.1%（P ＜ 0.01） and reduced plasma levels of arachidonic acid（by 44.2%; P = 0.02） and anandamide（by 49.7%; P = 0.03） postpartum compared to CTL. The m RNA transcription levels of the cannabinoid receptor 1（CNR1/CB1） tended to be lower（2.5 folds） in white blood cells of FLX than in CTL（P = 0.10）, and protein abundance of ECS enzyme monoacylglycerol lipase was higher in peripheral blood mononuclear cells of FLX than in CTL（P = 0.04）. In adipose tissue, palmitoylethanolamide levels were lower in FLX than in CTL（by 61.5%; P = 0.02）, relative m RNA transcription of lipogenic genes were higher, and the protein abundance of cannabinoid receptor 2（P = 0.08） and monoacylglycerol lipase（P = 0.10） tended to be higher in FLX compared to CTL. Hepatic 2-arachidonoylglycerol tended to be higher（by 73.1%; P = 0.07）, and interlukin-6 m RNA transcription level was 1.5 folds lower in liver of FLX than in CTL（P = 0.03）.Conclusions: Nutritional supplementation of omega-3 fatty acids seems to partly modulate ECS activation, which could be related to lower feed intake. The altered ECS components in blood, adipose tissue and liver are associated with moderate modulations in lipid metabolism in the adipose and inflammation in liver of peripartum dairy cows.