This study aimed to investigate the effects of trimethylamine N-oxide (TMAO) on vascular calcification (VC) in db/db mice induced by a high-phosphorus diet and to identify the associated molecular pathways. To establish a VC model, db/db mice were fed an adenine-supplemented high-phosphorus diet, with TMAO and its inhibitor 3,3-dimethyl-1-butanol (DMB) administered via drinking water. The effect of TMAO on renal pathology and colonic mucosal barrier function in db/db mice was evaluated. Micro-computed tomography (Micro-CT) and Von Kossa staining were used to evaluate the degree of aortic calcification. Western blotting, immunohistochemistry, immunofluorescence, and RT-qPCR were employed to detect the proteins and mRNAs related to the sirtuin 1 (SIRT1)/nuclear factor-κB p65 (NF-κB p65)/NOD-like receptor pyrin domain-containing 3 (NLRP3) signaling pathway. Sequencing of the 16S rRNA gene was performed to analyse changes in the gut microbiota. We found that the serum TMAO level in db/db-VC model mice was significantly elevated, accompanied by calcium and phosphorus metabolism disorders and gut microbiota imbalance, mainly manifested as an increase in the abundance of Prevotella and Desulfovibrio. TMAO aggravated renal dysfunction and intestinal barrier injury in db/db-VC mice and induced a certain degree of inflammatory response. Moreover, TMAO intervention significantly upregulated the expression of osteogenic marker proteins. In contrast, inhibition of TMAO production by DMB significantly reduced VC. Mechanistically, TMAO inhibited the activity of the SIRT1 deacetylase, promoted the acetylation of NF-κB p65, and subsequently activated its downstream NLRP3 inflammasome and IL-1β signaling.