Colon-targeted riboflavin supplementation produced distinct dose-dependent changes in gut microbiome composition and function across 348 healthy adults aged 50-70. The 10mg dose generated the most comprehensive microbial shifts, increasing alpha diversity, total species counts, and community resilience markers while enhancing riboflavin biosynthesis pathways. The 75mg dose specifically boosted butyrate production at week 4, while the lowest 1.4mg dose reduced dysbiosis indices. This targeted delivery approach represents a significant advancement over conventional B2 supplements, which are absorbed in the small intestine before reaching the colon where most gut bacteria reside. The microbiome modulation occurred without adverse effects on inflammatory markers or gut barrier integrity, suggesting therapeutic potential for age-related microbiome decline. However, the clinical significance of these microbial changes for healthspan or disease prevention remains unclear, as host health biomarkers showed minimal between-group differences. As a preprint awaiting peer review, these promising findings require validation through the formal review process. The research opens new possibilities for precision microbiome interventions using colon-specific nutrient delivery systems.