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• N-carbamylglutamate (NCG) improved the production performance of dairy cows. • NCG enhanced antioxidant capacity and immune function to ameliorate heat stress. • NCG reduced blood urea nitrogen and ammonia, while increased serum glucose. • Sixteen plasma metabolites related to heat stress were improved by NCG. • Pathway analysis revealed the potential mechanism of NCG on heat stress. Heat stress is a serious threat to dairy cow health worldwide. N-carbamylglutamate (NCG), a novel feed additive, has been applied in dairy cow industry. However, the effects and mechanisms of NCG on dairy cows under heat stress are still unknown. A total of 48 Holstein dairy cows with similar days in milk (154 ± 13.6 d), parity (1-3), and body condition score were randomly divided into 4 groups of 12 animals each. Under heat stress, the cows were fed a TMR diet supplemented with 0 (control), 15, 20, 25 g of NCG/d per cow for 60 days. Milk production performance was recorded and serum parameters were examined. Meanwhile, metabolomics study of plasma based on liquid chromatography-mass spectrometry was also applied to investigate metabolites and pathways in response to NCG supplementation. Milk yield was increased but milk urea nitrogen was reduced by the NCG treatment. Addition of 15 g of NCG/d increased the milk protein percentage compared with the control. No effect of NCG was observed on dry matter intake, lactose percentage, milk fat percentage and somatic cell count. Serum glucose concentrations in cows fed with 15, 20, 25 g of NCG/d were 5.34 ± 0.312, 5.78 ± 0.473 and 5.54 ± 0.711 mmol/L, which were greater than those in the control (4.87 ± 0.367 mmol/L, P < 0.05). Meanwhile, the concentrations of blood urea nitrogen and blood ammonia were decreased with the addition of NCG. Results for antioxidant capacity and immune function showed beneficial effects of NCG, such as the increases in total antioxidant capacity, superoxide dismutase, glutathione peroxidase, IgG, IgM and IgA. Clear separations of plasma metabolic profiles between control and NCG groups were observed in the score plots. Sixteen different metabolites involved in metabolisms of amino acids, ketone bodies, butanoate and energy, as well as gut microbiome-derived metabolism were regulated by NCG supplementation. In conclusion, supplementation with NCG has the potential to enhance production performance, antioxidant capacity and immune function, and improved the metabolic profile of dairy cows under heat stress. [ABSTRACT FROM AUTHOR]