Background Vit B12 deficiency is a recognized complication of ‘prolonged metformin therapy in patients with Type 2 Diabetes Mellitus (T2DM). This deficiency can contribute to neuropathy, anemia, and cognitive dysfunction, often overlapping with diabetic complications’. Despite its clinical significance, routine screening for ‘vit B12 levels in diabetic patients on metformin remains infrequent’. This study aims to evaluate the prevalence of vit B12 deficiency and its associated risk factors in patients receiving metformin therapy. Methods A cross-sectional study was conducted at Jinnah Medical College, Peshawar, from April 2023 to April 2024. A total of 103 patients with T2DM on metformin therapy were enrolled. Data on demographics, clinical history, metformin duration and dosage, gastrointestinal symptoms, and neuropathy signs were collected. Blood samples were analyzed for ‘serum vit B12 levels, methylmalonic acid (MMA), homocysteine, folate, and mean corpuscular volume (MCV). Patients were classified as having normal (>300 pg/mL), borderline (200-300 pg/mL), or deficient (<200 pg/mL) vit B12 levels’. Statistical analysis was performed to determine significant associations between vit B12 status and various clinical factors. Results ‘The study found that 36.9% of patients had vit B12 deficiency, while 34.0% had borderline levels, indicating that over 70% of participants had suboptimal B12 levels’. A significant association was observed between long-term metformin use (>10 years) and higher doses (>2000 mg/day) with low vit B12 levels (p<0.05). Patients with neuropathy symptoms and gastrointestinal complaints were more likely to be vit B12 deficient. Elevated MMA and homocysteine levels were also observed in patients with low serum vit B12. Additionally, Helicobacter pylori infection, chronic gastritis, and vegetarian diets were significantly linked to lower vit B12 levels (p<0.05). Conclusion This study highlights a high ‘prevalence of vit B12 deficiency in diabetic patients on metformin therapy, particularly among those with long-term use, higher dosages, and gastrointestinal disorders’. Given the overlap between neuropathy symptoms and B12 deficiency, routine screening of vit B12 levels in diabetic patients on prolonged metformin therapy is essential. Early detection and timely supplementation can help prevent long-term complications and improve patient outcomes.