BACKGROUND: Trimethylamine N-oxide (TMAO) is a gut microbial metabolite that promotes calcified aortic valve disease (CAVD), but the underlying mechanism remains obscure. Herein, we aim to test the hypothesis that TMAO regulated the inflammatory process in aortic valves via N6-methyladenosine (m6A) RNA methylation-mediated macrophage polarization. METHODS: In vitro, we stimulated macrophages (Phorbol-12-Myristate-13-Acetate-induced THP-1) with TMAO and assessed the expression of methyltransferase-like 3 (Mettl3), IL-1 receptor associated kinase M (IRAK-M) and polarization markers. The interaction between YTH domain family protein 2 (YTHDF2) and IRAK-M mRNA was explored by RNA-IP and RNA decay assay. Functionally, the effects of macrophages on human aortic valve interstitial cells (AVICs) were measured via macrophage adhesion assay and co-culture system. In vivo, the influence of IRAK-M on CAVD development was verified using Irak-m RESULT: Mettl3 was highly expressed while IRAK-M was decreased in human calcified aortic valves. In vitro, TMAO upregulated the expression of Mettl3, while the expression of IRAK-M, an important negative regulator of the NF-κB pathway, was remarkably decreased in macrophages. TMAO also induced classical macrophage activation (M1 polarization). Mechanistically, IRAK-M was identified as a target of Mettl3-mediated m6A modification, indicating the involvement of m6A methylation in the regulation of NF-κB activation. Moreover, RIP assay revealed the direct interaction between YTHDF2 and IRAK-M mRNA and this process was dependent on Mettl3. TMAO-treated macrophage conditioned medium induced inflammatory responses in human aortic valve interstitial cells (AVICs). In vivo experiments showed that the deletion of IRAK-M significantly accelerated the progression of aortic valve lesion in mice administrated with high-fat and choline diet (HFCD). CONCLUSION: TMAO induces the expression of Mettl3 in macrophages. Mettl3 promotes M1 polarization of macrophages by inhibiting IRAK-M through a m6A/YTHDF2 pathway. TMAO-treated macrophages aggravate the inflammation of human AVICs.