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Aim: We investigated cross-sectional associations between circulating homocysteine, folate, biomarkers of vitamin B 12 status and brain volumes. We furthermore compared brain volumes of participants who received daily folic acid and vitamin B 12 supplementation with participants who did not., Methods: Participants of the B-PROOF study ( n = 2919) were assigned to 400 µg folic acid and 500 µg vitamin B 12 , or a placebo. After two years of intervention, T₁-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 B 12 , holotranscobalamin, and methylmalonic acid concentrations were measured., Results: Multiple linear regression analyses showed inverse associations between plasma homocysteine with TBV (β = -0.91, 95% CI -1.85-0.03; p = 0.06) and between serum folate and TBV (β = -0.20, 95% CI -0.38, -0.02; p = 0.03). No significant associations were observed for serum vitamin B 12 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 B 12 supplementation will be beneficial above and beyond normal dietary intake for brain health., Competing Interests: The authors declare no conflict of interest.