Folate, vitamin B6, and vitamin B12 intake can be important regulators for obesity development. Thus, we investigated the possible association between the intake of these vitamins and the excess body weight or obesity prevalence in the participants of the Cohort of Universities in Minas Gerais (CUME project). This study analyzed cross-sectional data of 2695 graduates and postgraduates from universities in the state of Minas Gerais (801 men, 1894 women, ages 36.2 ± 9.4). The first step consisted of collecting data online, and the second step consisted of blood collecting in the subsample living in the city of Viçosa and its region (Minas Gerais). Excess body weight and obesity prevalence were 38.1% and 10.1%, respectively. Inadequate intake of folate, B6, and B12 were 12, 6.3, and 11.1%, respectively. Beans/lentils and French bread presented the highest contribution to folate intake (23.45% and 10.01%, respectively). Those individuals in the third tertile for folate intake (≥511.12 μg/d) had a lower excess body weight [prevalence ratio (PR): 0.79, confidence interval (CI): 0.71–0.8] and obesity prevalence (PR: 0.60, CI: 0.45–0.78). These associations were maintained when the sample was categorized by sex. In addition, serum folate was positively associated with dietary folate (p for trend = 0.032) and negatively associated with serum homocysteine (p for trend = 0.003) in the subsample. Dietary folate intake was negatively associated with excess body weight and obesity in CUME participants, indicating the relevance of this vitamin dietary assessment.