Long term administration of metformin interferes with the absorption of vitamin B12 resulting in vitamin B12 deficiency.Among the various genetic variants that are associated vitamin B12 deficiency, we analyzed BHMT gene polymorphism and their association with metformin induced vitamin B12 deficiency in T2DM patients.A cross sectional study was done with 300 participants. Methyl malonic acid, homocysteine and high sensitive C reactive protein were analysed by Mass Spectrometry, Chemiluminescent analyser and Immunoturbidimetric method respectively. Genetic variants were analysed by ARMS-PCR method, Data was analyzed with various statistical tools like ROC, Odds ratio and Likelihood ratio. Significant reduction in folic acid and vitamin B12 in metformin users was found. High sensitive C reactive protein, homocysteine and methyl malonic acid are significantly increased in patients with metformin induced B12 deficiency. ‘A’ allele in BHMT (A allele OR =2.1, AA genotype =2.8) showed risk of vitamin B12 deficiency in T2DM patients on metformin therapy. BHMT gene polymorphism had LR of 2.65 for folic acid (AG genotype), 2.73 for MMA (AA genotype) and 2.63 for Homocysteine (AA genotype).We found an association between single nucleotide polymorphism of BHMT and diagnosis of vitamin B12 deficiency status in metformin users. Folic acid, MMA and homocysteine had high specificity in concordance with BHMT (rs3733890) polymorphism in predicting vitamin B12 deficiency. Early screening of SNP of BHMT in T2DM patients on metformin therapy will help us to identify group of people who are prone for vitamin B12 deficiency.