Obesity is considered to be one of the most prevalent diseases in modern-day society. It is an illness with complex etiology that occurs due to an imbalanced input and usage of energy where the input of energy is larger than the usage. Obesity is the cause of many complications related to different organ systems. Vitamin B12 is a key vitamin in erythropoiesis, and methylation, and it is a cofactor of different enzymes. 80-94% of total cobalamin is an inactive fraction bonded to haptocorrin while the rest is bonded in holotranscobalamin. Vitamin B12 deficiency can lead to hematological abnormalities and demyelination of the central nervous system which is why early detection is important. Cobalamin concentration within the reference range does not equate to an adequate status of vitamin B12 which is why this assay is not the best marker of vitamin B12 deficiency. As an active form of cobalamin, the holotranscoobalamin assay represents a better choice in detecting deficiency. The goal of this research was to determine whether there is a statistically significant difference in cobalamin and holotranscobalamin concentrations between a group of obese subjects (BMI > 25 kg/m2) and a group of subjects with a normal body mass index. The research was conducted at the Department of Laboratory Diagnostics at the University Hospital Centre in Zagreb on 195 subjects, of which 103 had normal body mass index, and 92 were obese. Analytes concentrations were measured using the chemiluminescent microparticle immunoassay (CMIA) on the Abbott Alinity analyzer for cobalamin, and on the Architect analyzer for holotranscobalamin. The Mann-Whitney test was used to determine whether there was a statistically significant difference in the concentrations. The results show statistically significant lower cobalamin concentration in obese subjects compared to subjects with a normal body mass index. The median of measured cobalamin concentrations was not below the recommended cut-off value (150 pmol/L). Even though holotranscobalamin concentration measured in obese subjects was lower, there was not a statistically significant difference between the two groups of subjects.