Introduction: Metformin is the most widely administered anti-diabetic medication among type 2 diabetes mellitus (T2DM) patients. However, metformin induces vitamin B12 mal-absorption which may increase the risk of vitamin B12 deficiency among T2DM patients. Aim: To determine the prevalence of vitamin B12 deficiency among diabetic patients on metformin therapy. Materials & Methods: This is a case-control hospital-based study conducted in Khartoum state. A hundred subjects were involved, fifty were with type 2 diabetes patients underuse of metformin, and fifty were diabetic patients not underuse of metformin. Serum Vitamin B12 levels were estimated by the ELIZA method in a fully Automated Biochemistry Analyzer (Cobas). Results: Table 3.2 shows the mean serum level of vitamin B12 in diabetes underuse of metformin and control group respectively were (252.6±101.3, 339.4±112.3), there was a significant decrease of serum level of vitamin B12 in patients under treatment with metformin compared to patients not underuse of metformin (p value=0.000). Conclusion: Significant decrease of serum vitamin B12 in the diabetic patients under treatment with metformin compared to diabetic patients not use of metformin. Vitamin B12 deficient subjects presented with significantly longer duration of T2DM, duration of metformin use, and use of higher metformin doses compared to non-deficient subjects.