Background: Overweight and obesity (OO) are significant public health issues, and many elements, including genetics, epigenetics, sedentary lifestyle, comorbid conditions, psychological and environmental pressures, have been linked to OO. More than 2 billion people are presently impacted by the global obesity epidemic, which is still advancing relentlessly. It is a significant public health concern and a major contributor to healthcare costs, because it increases the chance of developing conditions such as heart disease, stroke, type 2 diabetes, and chronic kidney disease (CKD). Using the ranges of 18.5–25 for normality, 25–30 for overweight, and 30 for obesity, BMI (in kg/m2) is used to identify obesity. Vitamin deficiency is one of the causative factors associated with the increasing trend of obesity. Altered vitamin B12 status is a multifactorial trait; changes in B12 status are produced by several single nucleotide polymorphisms (SNPs) in various genes that interact with the environment. They also support coordinated efforts to alter the built environment that is causing the obesity pandemic. Therefore, the present study aimed to evaluate theTCN-2(776C>G) gene alteration and vitamin B12 levels with respect to different body mass index, as well as associating BMI with other biochemical parameters. Methods: 250 individuals were involved in the study; among them, 100 were in the healthy weight range category (BMI: 18.5 to <25 kg/m2), 100 were overweight (BMI: 25.0 to <30 kg/m2), and 50 were obese (BMI: >30 kg/m2). Participants visited during the screening program were subjected to blood pressure measurement, and further peripheral blood samples were drawn from all the participants in plain as well as in EDTA vials for biochemical (lipid profile and vitamin B12 level) analysis and single nucleotide polymorphism studies. Extracted DNA from whole blood collected in EDTA vials using kit protocol was used for genotyping by PCR-RFLP. Results: The levels of systolic (p< 0.0001) and diastolic blood pressures (p< 0.0001), HDL (p< 0.0001), LDL (p= 0.04), TG (p< 0.0001), cholesterol (p< 0.0001), and VLDL (p< 0.0001) showed significant differences between healthy controls, overweight, and obese groups. The healthy controlTCN-2(776C>G) genotypes were compared with those of overweight and obese participants, and compared to the healthy controls it was observed that overweight (p= 0.01) and obese (p= 0.002) subjects had significant differences inTCN-2(776C>G) genotypes. For genotypes CG and GG, the odds ratio was 1.61 (0.87–2.95;p= 0.12), and 3.81 (1.47–9.88;p= 0.005) for overweight participants, respectively, and obese participants’ calculated odds ratios were 2.49 (1.16–5.36;p= 0.01) and 5.79 (1.93–17.35;p= 0.001), respectively. The relative risk for genotypes CG and GG, was 1.25 (0.93–1.68;p= 0.12), 2.17 (1.12–4.17;p= 0.02) for overweight participants, while the obese participants’ calculated relative risks were 1.31 (1.03–1.68;p= 0.01) and 2.02 (1.12–3.65;p= 0.001), respectively. Vitamin B12 levels were analyzed, and it was observed that a significant difference existed among overweight (305.5 pmol/L,p< 0.0001) and obese patients (229 pmol/L,p< 0.0001), respectively, as compared to healthy controls (385.5 pmol/L). Correlation analysis showed a significant association of vitamin B12 level with TG, cholesterol and VLDL; it showed a negative correlation, suggesting that decreases in B12 levels may impact the lipid profile. Conclusion: The study concluded that a predisposition to the GG genotype ofTCN-2gene polymorphism (776C>G) may increase susceptibility to obesity and the related complications, and higher odds and relative risk for the GG genotype may increase the risk of having obesity and further related complications. Lower vitamin B12 levels were linked with obesity and overweight, and impaired lipid parameters suggested that lower vitamin B12 may impact the altered lipid profile.