Abstract: Objective To screen for specific metabolites in patients with large artery atherosclerosis (LAA) in the head and neck and to verify whether these metabolic markers can upregulate the NLR family pyrin domain containing protein 3 (NLRP3) inflammasome to mediate vascular inflammatory responses, thereby inducing the development of LAA in mice. Methods A total of 21 LAA patients and 18 healthy controls were included. Plasma non-targeted metabolomics analysis was performed to screen for specific metabolites. Male C57BL/6J mice were randomly divided into three groups (n=8): a sham surgery group (Sham), a trimethylamine-N-oxide (TMAO) treatment group, and a TMAO + 3,3-dimethylbutyrate (DMB) treatment group. The Sham group was fed a standard diet, the TMAO group was given 0.12% TMAO in drinking water and a high-fat diet, and the TMAO+DMB group was given 1% DMB in drinking water in addition to TMAO treatment. After 4 weeks, blood samples were collected to measure total cholesterol (TCH), low-density lipoprotein (LDL), and apolipoprotein B (ApoB) levels. Oil red O staining was used to assess the percentage of plaque area in the carotid arteries. The levels of NLRP3, cleaved caspase-1(cle-caspase-1), and cleaved IL-1β(cle-IL-1β) were detected to verify the pro-plaque formation effect of specific metabolites. Results Non-targeted metabolomics identified TMAO as a specific metabolite for LAA. Pearson correlation analysis showed that TMAO was positively correlated with TCH, LDL, and ApoB levels (P<0.05). In mouse experiments, the TMAO group showed significantly higher blood lipid levels than the Sham group, while the TMAO+DMB group had lower levels than the TMAO group (P<0.05). Oil red O staining revealed that arterial plaque area in the TMAO group significantly increased, in comparison with reduced area in the TMAO+DMB group (P<0.05). Western blot indicated that the expression of NLRP3 and other inflammation-related proteins was significantly up-regulated in the TMAO group but downregulated in the TMAO+DMB group (P<0.05). Conclusions Distinct specific metabolic markers, such as TMAO, exist in the plasma metabolite expression profiles of patients with head and neck LAA compared to healthy individuals. The gut microbiome metabolite TMAO can upregulate blood lipid levels and enhance the NLRP3 inflammasome-mediated inflammatory response, accelerating the occurrence and progression of atherosclerosis.