Background <p>Emerging studies suggests a potential link between serum trimethylamine N-oxide (TMAO) and obesity in adults. However, data on this association in children remain scarce. The present study aimed to investigate the association of TMAO and its precursors with childhood obesity and to evaluate whether such associations would be modified by basic clinical characteristics.</p> Methods <p>We conducted a case-control study involving 50 children with obesity and 50 children with normal weight aged 2–6 years. Serum TMAO and its precursors were quantified by ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Multivariate logistic regression models and Pearson correlation analyses were performed after controlling several confounding factors.</p> Results <p>Serum choline was inversely associated with childhood obesity (OR (95% CI) for highest vs. lowest quartile: 0.16 (0.04, 0.61), <i>p =</i> 0.007), whereas serum creatinine and carnitine showed positive correlations (ORs: 30.03 and 5.31, <i>p</i> &lt; 0.001 and <i>p</i> = 0.011 respectively). We also found that choline was negatively correlated with BMI (<i>p</i> = 0.038), whereas creatinine and carnitine were positively correlated with BMI (<i>p</i> &lt; 0.001 and <i>p</i> = 0.002, respectively). in addition, subgroup analyses based on sex, age groups, and maternal gestational diabetes mellitus status showed generally consistent results, with no significant interactions. No significant associations of betaine, TMA and TMAO with childhood obesity were observed (all <i>p</i> &gt; 0.05).</p> Conclusion <p>Higher serum choline levels were associated with lower odds of childhood obesity, while elevated creatinine and carnitine levels were associated with higher odds. These findings provide hints regarding the potential significance of these metabolites, which can be influenced by gut microbiota activity, diet, and host metabolism, in early-life obesity.</p> Clinical trial number <p>Not applicable.</p>