Background Vitamin B12 deficiency (<148 pmol/L) and insufficiency (<221 pmol/L) constitute a global health problem, particularly in low-income settings and among children, women of reproductive age, and the elderly. There have been few Latin American national surveys to examine the burden of vitamin B12 deficiency and vitamin B12 insufficiency. The 2012 Ecuadorian National Survey of Health and Nutrition is a nationally-representative complex survey of the Ecuadorian population and provides the first national estimate of vitamin B12 status in Ecuador. Methods Vitamin B12 was measured in 16,152 people from 10 to 59 years of age. Association of vitamin B12 status and levels with different socio-demographic and anthropometric correlates (sex, age, area, region, economic quintile, education level, ethnicity, and body mass index) was explored with univariate and multivariate linear and logistic regression models using survey procedures in SAS 9.4. Results The prevalence of vitamin B12 deficiency and vitamin B12 insufficiency in the overall Ecuadorian population was 5.4% and 27.4%, respectively. The burden was highest among pregnant women (21% and 55%). In the multivariate analyses, sex, age, region, education level, economic quintile, and ethnicity were significantly associated with vitamin B12 status. Men had higher odds of vitamin B12 insufficiency when compared to women (OR 1.5, 95% CI 1.35-1.66). Individuals living in the coastal regions had higher natural logarithmically transformed vitamin B12 concentrations (β=0.21, p<0.0001) and lower odds of vitamin B12 deficiency (OR: 0.56, 95% CI: 0.44-0.72) and insufficiency (OR: 0.48, 95% CI 0.41-0.57) when compared to people living in the highlands. Conclusions Vitamin B12 insufficiency is a significant health problem in Ecuador. Information is lacking to estimate the burden of vitamin B12 deficiency and vitamin B12 insufficiency for significant risk groups such as infants, children <10y, pregnant and lactating women, and the elderly. Prospective studies are needed to determine the association of total vitamin B12 insufficiency with adverse health outcomes in this population.