Background: Although it is known that health literacy (HL) plays an explanatory role in educational inequalities in health, it is unknown whether this role varies across age groups. Objective: The purpose of this study was to investigate whether the mediating role of HL in educational inequalities in four health outcomes varies across age groups: age 46 to 58 years, age 59 to 71 years, and age 72 to 84 years. Methods: We used data from the Dutch Doetinchem Cohort Study, which included 3,448 participants. We included years of education as predictor, chronic illness prevalence and incidence, mental and self-perceived health as outcomes, and HL, based on self-report, as mediator. We used multiple-group mediation models to compare indirect effects across age groups. Key Results: In the complete sample without age stratification, HL partly mediated the effect of education on all health outcomes except for incidence of chronic diseases. These indirect effect estimates were larger for subjective (self-perceived health, proportion mediated [PM] = 37%, and mental health, PM = 37%) than for objective health outcomes (prevalence of chronic disease, PM = 17%). For the prevalence of chronic disease, the indirect effect estimate was significantly larger among individuals age 46 to 58 years compared to individuals age 59 to 71 years and for incidence of chronic disease also compared to individuals age 72 to 84 years. All other indirect effect estimates did not differ significantly between age groups. Using an alternative cut-off point for HL or adjusting for cognitive functioning did not meaningfully change the results. Conclusions: Overall, we found that the explanatory role of HL in educational inequalities in mental and subjective health was stable but that it varied across age groups for chronic diseases, where it was largest among individuals age 46 to 58 years. Future studies may investigate the benefits of starting to intervene on HL from a younger age but means to improve HL may also benefit the subjective health of ...