Background <p>This study aims to explore the causal relationship between gut microbiota and childhood obesity and to assess the potential mediating role of blood metabolites in this relationship.</p> Methods <p>This study covering 473 gut microbiota and 1400 metabolites. The bidirectional two-sample Mendelian Randomization method is employed, with Inverse Variance Weighted as the main statistical approach, to assess the causal relationships between gut microbiota, metabolites and Childhood Obesity.</p> Results <p>The study found significant causal associations between 12 types of gut microbiota and childhood obesity, with 7 microbiota showing a negative correlation and 5 acting as risk factors for obesity. Bacillaceae A showed the strongest association with childhood obesity (OR = 0.0481, <i>P</i> = 0.0126), while Eubacterium Q was identified as a major risk bacterium for obesity (OR = 5.4330, <i>P</i> = 0.0452). Additionally, 18 metabolites were found to be associated with childhood obesity, with 6-bromotryptophan being the strongest negatively correlated metabolite (OR = 0.5680, <i>P</i> = 0.00023) and methylsuccinate being the strongest positively correlated risk metabolite (OR = 1.6987, <i>P</i> = 0.0383). Mediation analysis identified 10 significant pathways, highlighting Adrenate (22:4n6) as a mediator for the gut microbiota UBA2922 sp900313925 in promoting childhood obesity (mediation effect: 0.106, mediation proportion: 10.94%), and X-12830 as a mediator for K10 sp001941205 in inhibiting childhood obesity (mediation effect: −0.0772, mediation proportion: 13.68%).</p> Conclusion <p>This study provides novel evidence of the causal roles of gut microbiota and blood metabolites in childhood obesity. The identification of key metabolites mediating the effects of gut microbiota on obesity risk offers potential targets for future interventions and therapeutic strategies.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>Analysis revealed 12 gut microbiota causally related to childhood obesity, with seven negatively correlated and five identified as risk factors. Bacillaceae A (protective) and Eubacterium Q (risk) were notably associated.</p> </ItemContent> <ItemContent> <p>Among 1400 metabolites studied, 18 showed causal relationships with obesity, notably 6-bromotryptophan (protective) and methylsuccinate (risk factor).</p> </ItemContent> <ItemContent> <p>Mediation analysis identified 10 significant causal pairings between gut microbiota and blood metabolites, with Adrenate (22:4n6) mediating the promotion of childhood obesity by UBA2922 sp900313925, and X-12830 mediating the inhibition of childhood obesity by K10 sp001941205.</p> </ItemContent> </UnorderedList></p>