It is known that the metabolism of homocysteine (Hcy) depends on the vitamins B6, B12 and folate, and furthermore that metformin reduces serum vitamin B12 levels. In order to investigate whether metformin treatment affects serum total Hcy (tHcy) levels we performed an open, prospective, randomised study in 60 non-diabetic male patients with cardiovascular disease. After a 4-week run-in period with lovastatin 40 mg day-1, and diet and lifestyle advice, patients were randomised into two groups, both continuing the run-in treatment. One group received metformin up to 2000 mg day-1, whereas the control group got no additional treatment. After 12 and 40 weeks of metformin treatment, tHcy levels increased moderately but significantly by 7.2% (p < 0.05) and 13.8% (p < 0.05) in the metformin group relative to the control group, whereas serum vitamin B12 levels decreased by 13.4% (p < 0.0005) and 17.7% (p < 0.0005), respectively. Serum folate levels did not change after 12 weeks, but decreased by 8.0% after 40 weeks (p = 0.061) relative to the control group. Serum levels of total cysteine and methylmalonic acid (MMA) did not change. In conclusion, metformin treatment increased tHcy levels and decreased levels of vitamin B12 and folate. Since MMA levels were unchanged, it remains an open question whether the increase in tHcy levels is secondary to reduced vitamin B12 levels, folate levels or a combination of both.