Background/objectives <p>Diets enriched with live microbes offer multiple health benefits. This study aimed to investigate the relationship between dietary intake of live microbes and accelerated biological age, as well as to examine the mediating role of insulin resistance.</p> Methods <p>The study included 4,909 participants from the National Health and Nutrition Examination Survey (NHANES) conducted between 2007 and 2018. Dietary live microbes intake was assessed using a self-report questionnaire. Insulin resistance was measured using the HOMA-IR and plasma insulin levels. Biological age and accelerated aging were assessed using PhenoAge and PhenoAgeAccel. We performed weighted multiple linear regression, logistic regression, restricted cubic spline (RCS) analysis, mediation analyses, and interaction analyses to explore the relationships between dietary live microbe intake, insulin resistance, and accelerated aging.</p> Results <p>Higher intake of live dietary microbes, compared to lower intake, was associated with a slower rate of accelerated aging after full adjustment for confounders (β = 0.15; 95% CI: − 0.26 to − 0.04; <i>P</i> = 0.008). RCS curves indicated an L-shaped dose–response relationship (<i>P</i>-nonlinear = 0.0003). Insulin resistance-related metrics (HOMA-IR and insulin) partially mediated these effects, with mediation proportions ranging from 24.65% to 30.03%. Interaction analyses revealed no significant interaction between dietary live microbe intake and other stratification factors regarding phenotypic age.</p> Conclusions <p>The intake of dietary live microbes is nonlinearly associated with accelerated biological age. IR partially mediated the relationship.</p>