Elevated plasma total homocysteine (tHcy) is a risk factor for vascular disease but lowering tHcy with B-vitamins, including folate, has generally not reduced vascular events in secondary prevention trials. Elevated plasma S-adenosylhomocysteine (AdoHcy) concentration may be a more sensitive indicator of vascular disease than plasma tHcy. However, unlike tHcy, plasma AdoHcy did not correlate with folate concentration in one study indicating that folate supplementation may not lower AdoHcy. Our aim was to determine whether providing B-vitamin supplements to healthy older people with elevated tHcy (>13 μmol/l) affects plasma AdoHcy and S-adenosylmethionine (AdoMet) concentrations. Healthy older participants (n 276; ≥ 65 years) were randomised to receive a daily supplement containing folate (1 mg), vitamin B12 (500 μg) and vitamin B6 (10 mg), or placebo, for 2 years. Of these participants, we selected the first fifty participants in each treatment group and measured plasma AdoHcy and AdoMet. Plasma tHcy was 4·4 (95 % CI 3·2, 5·6; P < 0·001) μmol/l lower at 2 years in the vitamins group compared with the placebo group. At 2 years, there were no significant differences in plasma AdoMet (+4 % (95 % CI − 2, 11); P = 0·19), AdoHcy ( − 1 % (95 % CI − 10, 8); P = 0·61) or the AdoMet:AdoHcy ratio (0·22 (95 % CI − 0·04, 0·49); P = 0·10) between the two groups. In conclusion, B-vitamin supplementation of older people lowered plasma tHcy but had no effect on plasma AdoMet or AdoHcy concentration. If elevated plasma AdoHcy is detrimental, this may explain why B-vitamins have generally failed to reduce vascular events in clinical trials.