Background:Long-term therapy with metformin was shown to decrease the Vitamin B12 level and manifested as peripheral neuropathy.Aim:The aim of this study is to define the prevalence of Vitamin B12 deficiency in early Type 2 diabetic patients (duration ≤5 years or drug treatment ≤3 years) and the relationship among metformin exposure and levels of cobalamin (Cbl), folic acid, and homocysteine (Hcy) with severity of peripheral neuropathy.Methodology:This is a cross-sectional study involving randomly selected ninety patients (male 56, female 34) between age groups of 35 and 70 years, comparing those who had received >6 months of metformin (Group A) (n = 35) with those without metformin (Group B) (n = 35) and patients taking metformin with other oral hypoglycemic agent (Group C) (n = 20). Comparisons were made clinically, biochemically (serum Cbl, fasting Hcy, and folic acid), and with electrophysiological measures (nerve conduction studies of all four limbs). Comorbidities contributing to neuropathy were excluded from the study.Results:Group A patients (54.28%) were prone to develop peripheral neuropathy comparing Group B (28.57%) and Group C (35%). There was significantly low plasma level of Cbl in Group A (mean 306.314 pg/ml) than in Group B (mean 627.543 pg/ml) and Group C (mean 419.920 pg/ml). There was insignificant low-level plasma folic acid in Group A (16.47 ng/ml) than in Group B (16.81 ng/ml) and Group C (22.50 ng/ml). There was significantly high level of Hcy in Group A (mean 17.35 µmol/L) and Group C (mean 16.99 µmol/L) than in Group B (mean 13.22 µmol/L). Metformin users even for 2 years showed evidence of neuropathy on nerve conduction velocity though their body mass index and postprandial blood sugar were maintained. There was significant difference in between groups regarding plasma Cbl, folic acid, and Hcy level as significance level <0.05 in all three groups (F [2, 87] = 28.1, P = 0.000), (F [2, 87] = 7.43, P = 0.001), (F [2, 87] = 9.76, P = 0.000). Post hoc study shows significant (P < 0.05) lowering of Cbl and Hcy level in Group A (mean = 306.314, standard deviation [SD] = 176.7) than in Group C (mean = 419.92, SD = 208.23) and Group B (mean = 627.543, SD = 168.33).Discussion:Even short-term treatment with metformin causes a decrease in serum Cbl folic acid and increase in Hcy, which leads to peripheral neuropathy in Type 2 diabetes patients. A multicenter study with heterogeneous population would have increased the power of the study. We suggest prophylactic Vitamin B12 and folic acid supplementation or periodical assay in metformin user.