Background: Betel nut (BN) or areca nut (AN) chewing habits on its own or with other ingredients including chewing tobacco are highly prevalent in many South East Asian countries as well as among the migrants from these countries residing around the world. The major alkaloid arecoline in the BN has been found to carcinogenic and to be associated with a range of health risks, including negative effects on pregnancy outcomes. Pregnancy imposes stress on folate stores because of increased requirements for growth of maternal tissues, fetus, and placenta. Folate defi ciency during pregnancy is a major public health concern as is associated with many adverse health outcomes including neural tube defects, low birth weight, preterm birth, and delayed maturation of the nervous system, growth retardation, and megaloblastic anemia. Objective: To investigate any association between BN consumption and folate status among pregn ant women in rural Bangladesh. Materials and Methods: Data of 730 pregnant women aged 14-50 years from the Maternal and Infant Nutrition Intervention in Matlab (MINIMat) trial in Bangladesh were included in this study. Logistic regression analysis and analyses of covariance (ANCOVA) were used. Results were adjusted for potential confounders. Results and Interpretation: Two-third (63%) of the women consumed BN and 17% had folate defi ciency. The women who consumed BN combined with chewing tobacco were 2.57 times more likely to have folate defi ciency (OR=2.57; 95% CI=1.23-5.36; P=0.012;) and the women who consumed BN 2-3 times/day were 2.51 times more likely to develop folate defi ciency among users (OR=2.51; 95% CI=1.07-5.92; P=0.035). Mean serum folate levels were signifi cantly lower among BN users as compared to nonusers. Conclusion: The results suggest that betel nut consumption combined with chewing tobacco is associated with lower serum folate level and folate defi ciency among pregnant women in rural Bangladesh. Strategies are needed for prevention and control of betel nut consumption in order to prevent adverse health outcomes.