A prospective cohort study was conducted from 2009 to 2012 to assess the relationship between serum baseline 25-hydroxivytamin D (vitamin D) status and the incidence of tuberculosis (TB) among 572 contacts of 89 pulmonary TB patients in Castellon, Spain. Three new cases of pulmonary TB occurred, with an incidence density of 3.6 per 1000 person-years. Mean vitamin D status was 13.7 ng/ml for cases and 25.7 ng/ml for non-cases. Vitamin D status showed a significant inverse association with TB incidence (adjusted HR 0.88, 95%CI 0.80-0.97). This result is in line with the hypothesis that vitamin D deficiency is associated with TB incidence.