Background: Heat stress (HS) becomes a serious threat to dairy cow industry in certain circumstances, Ncarbamylglutamate (NCG) is a novel feed additive that may be used to alleviate HS. However, the effects and mechanisms of NCG on dairy cows under HS are still unknown. A total of 48 Holstein dairy cows with similar days in milk (154.37±13.56 d), parity (1-3), and body condition score (BCS) were randomly divided into 4 groups of 12 animals each. Under HS, 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 (LC-MS) was also applied to investigate metabolites and pathways in response to NCG supplementation.Results: Milk yield (MY) was increased but milk urine nitrogen (MUN) was reduced by the NCG treatment.Addition of 15 g of NCG/d increased the milk protein percentage (MPP) compared with the control. No effect of NCG was observed on dry matter intake (DMI), lactose percentage (LP), milk fat percentage (MFP) and somatic cell count (SCC). Serum glucose (GLU) levels in cows fed with 15, 20, 25 g of NCG/d were increased by 14.35%, 19.34% and 18.63% in comparison with the control. Meanwhile, the concentrations of blood urea nitrogen (BUN) and blood ammonia (BA) were decreased with the addition of NCG. Results for antioxidant capacity and immune function showed bene cial effects of NCG, such as the increases in total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), IgG, IgM and IgA. Clear separations of plasma metabolic pro les 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.Conclusions: This study found that NCG treatment improved antioxidant capacity, immune function, production performance, and metabolic pro le of dairy cows under HS and provided new evidence for the better utilization of NCG.