Long-term and high-dose treatment with metformin is known to be associated with vitamin B12 deficiency in patients with type 2 diabetes. We investigated whether the prevalence of B12 deficiency was different in patients treated with different combination of hypoglycemic agents with metformin during the same time period. A total of 394 patients with type 2 diabetes treated with metformin and sulfonylurea (S+M group, n = 299) or metformin and insulin (I+M group, n = 95) were consecutively recruited. The vitamin B12 and folate levels were quantified using the chemiluminescent enzyme immunoassay. Vitamin B12 deficiency was defined as vitamin B12≤300 pg/mL without folate deficiency (folate>4 ng/mL). The mean age of and duration of diabetes in the subjects were 59.4±10.5 years and 12.2±6.7 years, respectively. The mean vitamin B12 level of the total population was 638.0±279.6 pg/mL. The mean serum B12 levels were significantly lower in the S+M group compared with the I+M group (600.0±266.5 vs. 757.7±287.6 pg/mL, P<0.001). The prevalence of vitamin B12 deficiency in the metformin-treated patients was significantly higher in the S+M group compared with the I+M group (17.4% vs. 4.2%, P = 0.001). After adjustment for various factors, such as age, sex, diabetic duration, duration or daily dose of metformin, diabetic complications, and presence of anemia, sulfonylurea use was a significant independent risk factor for B12 deficiency (OR = 4.74, 95% CI 1.41-15.99, P = 0.012). In conclusion, our study demonstrated that patients with type 2 diabetes who were treated with metformin combined with sulfonylurea require clinical attention for vitamin B12 deficiency and regular monitoring of their vitamin B12 levels.