ObjectiveTo assess the prevalence of vitamin B12 deficiency in people with type 2 diabetes mellitus (T2DM) on metformin and without metformin.MethodologyBetween May 2018 and January 2019, this prospective multicenter observational study recruited participants from seven centers in four provinces of Pakistan (Sindh, Punjab, Baluchistan and Khyber Pakhtunkhwa). Participants with T2DM treated with metformin for >2 years and those not on metformin underwent assessment of hemoglobin, vitamin B12, homocysteine and diabetic neuropathy (vibration perception threshold (VPT) >15V) and painful diabetic neuropathy (Douleur Neuropathique 4 (DN4) ≥4) and Diabetic Neuropathy Symptom (DNS) score ≥1.ResultsOf 932 subjects, 645 (69.2%) were treated with metformin, while 287 (30.8%) were not on metformin. Overall, B12 deficiency (<200 pg/mL) was significantly higher in metformin users of 25 (3.9%), compared with non-metformin users of 6 (2.1%), while B12 insufficiency (200–300 pg/mL) was significantly lower in metformin users of 117 (18.4%) compared with non-metformin users of 80 (27.9%). Subjects with B12 deficiency and insufficiency with hyperhomocysteinemia (≥15) were found in 19 (76%) µmol/L and 69 (60.5%) µmol/L in metformin users compared with 6 (100%) µmol/L and 57 (73.1%) μmol/L in non-metformin users, respectively. VPT>25 and DN4 score ≥4 were significantly higher in B12-deficient metformin users compared with non-metformin users. Similarly, DNS score ≥1 was non-significantly higher in B12-deficient metformin users compared with non-metformin users.ConclusionThis study shows that vitamin B12 insufficiency was frequently found in our population and may progress into B12 deficiency. It is also associated with neuropathy in subjects on metformin. Further interventional studies to assess the benefit of B12 treatment on painful neuropathy in patients on metformin may be warranted. B12 levels may be checked in people with T2DM using metformin for >2 years.