BACKGROUND: Vitamin B12 deficiency is associated with many adverse health outcomes and is highly prevalent worldwide. The present study assesses the prevalence and socio-demographic factors associated with vitamin B12 deficiency in a representative sample of pregnant women in Colombia.\n\nMETHOD: We used data from the cross-sectional, nationally representative survey (ENSIN, 2010). A total of 1.781, (13-49 years old) pregnant women were enrolled. Serum Vitamin B12 a concentration was determined by chemiluminescence and sociodemographic date was assessed by computer-assisted personal interview technology. Multivariate analyses using unordered multinomial logistic regression models were conducted in the main analysis.\n\nRESULTS: Vitamin B12 concentrations ranged from 45 to 1000 pg/mL (mean 299.2 pg/mL, 95% CI 290.6 to 303.7 pg/mL). A total of 18.6% of pregnant women had vitamin B12 concentrations below 200 pg/mL and 41.3% had concentrations between 200 and 300 pg/mL. Being of indigenous ethnicity, living in the east and living in a rural area showed the lowest mean values (273.2 pg/mL, 270.8 pg/mL and 290.1 pg/mL, respectively). The multivariate logistic regression shows that pregnant women belonging to the indigenous ethnic group OR 2.2, (95% CI 1.1 to 4.3), living in the pacific region (west) OR 4.4, (95% CI 2.8 to 6.9), or national territories (south) OR 2.3, (95% CI 1.4 to 3.7) were associated with a higher probability of serum vitamin B12 deficiency.\n\nCONCLUSION: The prevalence of vitamin B12 deficiency in Colombian pregnant women is substantial. Factors associated with depletion among pregnant women should be considered for future interventions in countries experiencing nutritional transition.