Elevated homocysteine (Hcy) levels (≥15 μmol/L) in the elderly are frequently associated with a higher risk of cardiovascular disease and cognitive decline. Several studies have already shown an Hcy-lowering effect of B vitamin supplementation in cohorts deficient in these nutrients. The aim of this randomized, double-blinded 12-week intervention study was to investigate whether Hcy levels in healthy elderly subjects (75.4±4.5 years, n=133) could be lowered with a micronutrient supplement (i.e., 400 μg folic acid, 100 μg cobalamin). Difference in mean initial Hcy levels between intervention (17.6±7.1 μmol/L, n=65) and placebo group (18.9±6.1 μmol/L, n=68) was not significant. The prevalence of cobalamin and folate deficiency in the total study population was low: 27% had serum-cobalamin levels ≤150 pmol/L, 12% holo-transcobalamin (Holo-TC) levels ≤50 pmol/L, 13% low cobalamin status using the aggregated cobalamin marker 4cB12 and 10% red blood cell (RBC) folate ≤570 nmol/L. Nevertheless, the treated subjects still showed improved cobalamin and folate biostatus (serum cobalamin Δt(12)-t(0): 63±48 pmol/L; Holo-TC Δt(12)-t(0): 17±19 pmol/L; RBC folate Δt(12)-t(0): 326±253 nmol/L) and Hcy levels (Δt(12)-t(0): -3.6±5.7 μmol/L). The effects were statistically significant compared to the placebo group with p=0.005 (serum cobalamin), p=0.021 (Holo-TC), p=0.014 (RBC-folate) and p<0.001 (Hcy). The Hcy-lowering effect was dependent on the initial Hcy levels (p<0.001). Our findings suggest that elevated Hcy levels in elderly subjects can be lowered regardless of the initial cobalamin and folate biostatus.