AIM: We investigated cross-sectional associations between circulating homocysteine, folate, biomarkers of vitamin B12 status and brain volumes. We furthermore compared brain volumes of participants who received daily folic acid and vitamin B12 supplementation with participants who did not. METHODS: Participants of the B-PROOF study (n = 2919) were assigned to 400 microg folic acid and 500 microg vitamin B12, or a placebo. After two years of intervention, T(1)-weighted magnetic resonance imaging (MRI) scans were made in a random subsample (n = 218) to obtain grey and white matter volume, and total brain volume (TBV). Plasma homocysteine, serum folate, vitamin B12, holotranscobalamin, and methylmalonic acid concentrations were measured. RESULTS: Multiple linear regression analyses showed inverse associations between plasma homocysteine with TBV (beta = -0.91, 95% CI -1.85-0.03; p = 0.06) and between serum folate and TBV (beta = -0.20, 95% CI -0.38, -0.02; p = 0.03). No significant associations were observed for serum vitamin B12 and holotranscobalamin. Fully adjusted ANCOVA models showed that the group that received B-vitamins had a lower TBV (adjusted mean 1064, 95% CI 1058-1069 mL) than the non-supplemented group (1072, 95% CI 1067-1078 mL, p = 0.03). CONCLUSIONS: Results were contradictory, with higher Hcy levels associated with lower TBV, but also with higher folate levels associated with lower TBV. In addition, the lack of a baseline measurement withholds us from giving recommendations on whether folic acid and vitamin B12 supplementation will be beneficial above and beyond normal dietary intake for brain health.