Vitamin B12 deficiency is associated with an increased risk of pregnancy complications and adverse birth outcomes. However, data on vitamin B12 deficiency in pregnant Bangladeshi women are limited. This study examines vitamin B12 deficiency and marginal deficiency in rural Bangladeshi women during early and late pregnancies. Some 522 women whose gestational age was <20 weeks were recruited. Serum vitamin B12 concentrations were measured at baseline and after 14 weeks of iron-folate supplementation. Logistic regression analysis examined the association of various socio-demographic, dietary, and pregnancy-related factors with vitamin B12 deficiency and marginal deficiency. Overall, 19% of the women during early pregnancy had vitamin B12 deficiency (serum vitamin B12 concentration < 203 pg/mL) and nearly 40% had marginal deficiency (serum vitamin B12 concentration 203 to <300 pg/mL). Vitamin B12 deficiency doubled to 38% during late pregnancy, while marginal deficiency slightly increased to 41.7%. The pregnant women with a gestational age of ≥27 weeks had a higher risk of developing vitamin B12 deficiency (OR = 2.61; 95% CI = 1.096–6.214) than those of a gestational age of <27 weeks. Vitamin B12 deficiency was significantly higher in pregnant women in rented accommodation (OR = 13.32; 95% CI = 1.55–114.25) than in those living in their own house. Vitamin B12 deficiency was significantly higher among women who consumed red or organ meat <3 times a week than in those who consumed it more often (OR = 2.327, 95% CI = 1.194–4.536). None of these factors were significantly associated with marginal vitamin B12 deficiency. In conclusion, vitamin B12 deficiency and marginal deficiency among pregnant rural Bangladeshi women increased as their pregnancies progressed. Increasing gestational age, living in a rented house, and the consumption of red or organ meat <3 times a week were identified as the independent risk factors of vitamin B12 deficiency in this population. Further research with more in-depth assessments of dietary vitamin B12 intakes is needed to develop an intervention program preventing vitamin B12 deficiency in this population.