AbstractObesity predicts adverse microvasculature from childhood, potentially via inflammatory pathways. We investigated whether inflammation mediates associations between obesity and microvascular parameters. In 1054 children (mean age 11 years) and 1147 adults (44 years) from a cross-sectional study, we measured BMI (z-scores for children) and WHtR, Glycoprotein acetyls (GlycA), an inflammatory marker, and retinal arteriolar and venular calibre. Causal mediation analysis methods decomposed a “total effect” into “direct” and “indirect” components via a mediator, considering continuous and categorical measures and adjusting for potential confounders. Compared to normal-weight BMI children, those with overweight or obesity had narrower arteriolar calibre (total effects −0.21 to −0.12 standard deviation (SD)): direct (not mediated via GlycA) effects were similar. Children with overweight or obesity had 0.25 to 0.35 SD wider venular calibre, of which 19 to 25% was mediated via GlycA. In adults, those with obesity had 0.07 SD greater venular calibre, which was completely mediated by GlycA (indirect effect: 0.07 SD, 95% CI −0.01 to 0.16). Similar findings were obtained with other obesity measures. Inflammation mediated associations between obesity and retinal venules, but not arterioles from mid-childhood, with higher mediation effects observed in adults. Interventions targeting inflammatory pathways may help mitigate adverse impacts of obesity on the microvasculature.