Abstract Background Myocardial infarction is the major cause of deaths worldwide. Gut bacteria can process choline as abundant in red meat and subsequently converted by flavin containing monooxygenase in the liver to trimethylamin-N-oxide (TMAO) metabolite, which is strongly associated with cardiovascular events. Aim To investigate the gut microbiome and its association with atherosclerotic plaque instability. Methods Forty-eight Apolipoprotein E deficient mice were randomly divided into two groups and two time points, fed with a high fat diet (containing either 0.4% choline or 3% choline) at 12 weeks of age, for 7 weeks or for 14 weeks. All mice underwent Tandem Stenosis (TS) surgery to induce the development of unstable plaques. Stool samples were collected directly from the colon. Measurements of gut microbes were performed by AGRF diversity profiling. After bacterial genomic DNA isolation, 16S rRNA were sequenced by targeting 27F-519R (V1-V3) and 341F-806R (V3-V4) on the Illumina MiSeq platform. Vessel segments of TS were histologically processed and plaque composition of lipid, collagen, and intraplaque hemorrhage (marker of unstable plaques) were performed by a series of chemical staining and immunohistochemistry. Results Monocytes and granulocytes in mouse blood were significantly increased in the high choline group (p<0.05, unpaired t-test) after 7 weeks of high fat diet (21% fat, 0.15% Cholesterol, 3% Choline). Profiling of gut microbiota showed that Fimicutes were down regulated in the high choline group (p<0.05, unpaired t-test). Within Phylum Fimicutes, only Clostridia (class) Clostridiales (order) were significantly downregulated. Interestingly, histological analysis of TS segments showed that TER-119 (intraplaque haemorrhage marker) and CD42c (platelet marker) were significantly increased in the high choline group, indicating atherosclerotic plaques are more unstable and prone to rupture (p<0.05, unpaired t-test). Nevertheless, CD68 (Foam cells) in plaques, and total atherosclerotic plaque burden in the aortic sinus and aortic arch were not affected by the elevated levels of choline consumption. Conclusion Choline intake increases circulating monocytes and granulocyte numbers in the blood but not in the atherosclerotic plaque itself. Whereas the total plaque burden is not changed by an increased choline intake, the reduction of Fimicutes, Clostridia and Clostridiales seems to contribute to atherosclerotic plaque instability. Acknowledgement/Funding Heart Foundation 2018 Future Leader Fellowship (2018 FLF) ID: 102068 Chen