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Abstract Background and aims Recent studies have shown that trimethylamine N-oxide (TMAO) is a risk factor for cardiovascular disease (CVD) in different clinical settings, but few studies confirmed the association in a community-based general population. Methods This is a nested case-control study from a prospective cohort design. A total of 86 newly diagnosed CVD cases with a median follow-up period of 4.83 years and 86 matched controls were selected for the present analysis. Results Using the LC-MS/MS assays, we found that new CVD cases had a higher baseline levels of TMAO than controls [median (inter-quartile): 1.57 (0.79–2.29) μmol/L v.s 0.68 (0.23–1.40) μmol/L, p < 0.001]. After multivariable adjustment, individuals with TMAO ≥1.89 μmol/L (Q4) and 1.05–1.89 μmol/L (Q3) had odds ratio (OR) for CVD of 2.735 [95% confidence interval (CI): 1.328–5.630] and 2.544 (95% CI: 1.251–5.172) with the lowest quartile (<0.43 μmol/L) as reference. In addition, comparisons of areas under receiver operator characteristics curves confirmed that a model including TMAO had a better discrimination than one without (0.732 vs. 0.664, p = 0.045). Conclusions In the community-based general population, there was a positive association between TMAO and future risk of CVD. Addition of TMAO improved the prediction of CVD beyond traditional risk factors. We recommend considering TMAO as a potential novel preventive target in the management of low-risk CVD adults. Graphical abstract Image 1 Highlights • TMAO has a dose-dependent relationship with risk of CVD in the general population. • Addition of TMAO significantly improved the predictive ability of CVD risk. • TMAO is a potential novel preventive target for low-risk CVD adults. [ABSTRACT FROM AUTHOR]