Objective: To investigate the association between metformin use and vitamin B12 deficiency in patients with Type 2 Diabetes Mellitus (T2DM) aged above 40 years. Methods: A cross-sectional study was conducted on 50 T2DM patients (35 metformintreated and 15 non-metformin-treated) aged above 40 years. Serum vitamin B12 levels were measured, and vitamin B12 deficiency was defined as levels 10 years was significantly associated with an increased risk of vitamin B12 deficiency (adjusted OR: 5.18, 95% CI: 1.16-23.12, p=0.031). Conclusion: Metformin use is significantly associated with vitamin B12 deficiency in T2DM patients aged above 40 years. Regular monitoring of vitamin B12 levels and consideration of vitamin B12 supplementation are recommended for T2DM patients receiving long-term metformin therapy.