AIMS/HYPOTHESIS: Patients treated with metformin exhibit low levels of plasma vitamin B(12) (B(12)), and are considered at risk for developing B(12) deficiency. In this study, we investigated the effect of metformin treatment on B(12) uptake and distribution in rats. METHODS: Sprague Dawley rats (n = 18) were divided into two groups and given daily subcutaneous injections with metformin or saline (control) for three weeks. Following this, the animals received an oral dose of radio-labeled B(12) ((57)[Co]-B(12)), and urine and feces were collected for 24 h. Plasma, bowel content, liver, and kidneys were collected and analyzed for B(12), unsaturated B(12)-binding capacity, and (57)[Co]-B(12). RESULTS: Three weeks of metformin treatment reduced plasma B(12) by 22% or 289 [47-383] pmol/L (median and [range]) (p = 0.001), while no effect was observed on unsaturated B(12)-binding capacity. Compared with controls, the amount of B(12) in the liver was 36% (p = 0.007) higher in metformin-treated rats, while the B(12) content in the kidney was 34% (p = 0.013) lower. No difference in the total amount of absorbed (57)[Co]-B(12) present in the tissues and organs studied was found, suggesting that metformin has no decreasing effect on the B(12) absorption. CONCLUSIONS/INTERPRETATION: These results show that metformin treatment increases liver accumulation of B(12), thereby resulting in decreases in circulating B(12) and kidney accumulation of the vitamin. Our data questions whether the low plasma B(12) observed in patients treated with metformin reflects impaired B(12) status, and rather suggests altered tissue distribution and metabolism of the vitamin.