Aims: Metformin is widely used oral hypoglycemic agent in patients with type 2 diabetes, but it may cause vitamin B12 deficiency. The objective of this study is to evaluate the prevalence of vitamin B12 deficiency and the associated factors with metformin use in patients with type 2 diabetes. Method: A total of 806 adults diagnosed with type 2 diabetes using metformin were consecutively enrolled from January 2012 to January 2013, and their clinical and laboratory data were investigated. Serum vitamin B12 and folic acid level were quantified by chemiluminescent enzyme immunoassay. Vitamin B12 deficiency was defined as serum vitamin B12 concentrations ≤ 300 pg/mL without folate deficiency (folate >4 ng/mL). Results: Mean age and diabetic duration were 59.1±10.8 years and 11.3±7.9 years, respectively. The prevalence of vitamin B12 deficiency in patients with type 2 diabetes treated with metformin was 9.43% (n=76), and mean serum vitamin B12 level was 662.5±257.1 pg/mL. The patients with vitamin B12 deficiency had a longer duration of metformin use (p<0.001), higher daily dose of metformin (p<0.001) than the patients without vitamin B12 deficiency. Mutivariate analysis revealed significant associations between vitamin B12 deficiency and duration of metformin use and total daily dosage. Compared with daily metformin dose of <1,000 mg, the adjusted odds ratios for metformin dose of 1,000 to 2,000 mg, and >2,000 mg were 5.4 (95% CI, 1.26-23.1) (p=0.023) and 12.7 (95% CI, 2.74-58.5) (p=0.001), respectively. Compared with metformin use of <36 months, the adjusted odds ratios for metformin use of 36 to 60 months, 60 to 120 months, and >120 months were 2.67 (95% CI, 1.2-6.1) (p=0.020), 6.22 (95% CI, 2.8-13.7) (p<0.001), and 10.3 (95% CI, 3.7-28.5) (p<0.001), respectively. Conclusion: Our study showed an increased risk of vitamin B12 deficiency in patients with type 2 diabetes who were treated with metformin, especially in higher dosage and long duration. More studies are needed for its clinical implications.