Objectives We aimed to estimate the prevalence of folate deficiency and contributing factors among pregnant women.Design A community-based, cross-sectional study.Setting Haramaya District, Eastern Ethiopia.Participants Four hundred and forty-six pregnant women participated in the study.Primary outcome measure The prevalence of folate deficiency and risk factors.Results Overall, the prevalence of folate deficiency was 49.3% (95% CI 44.6% to 54.1%). Pregnant women with iron deficiency anaemia were 2.94 times more likely to develop folate deficiency (adjusted OR (AOR)=2.9, 95% CI 1.9 to 4.7). Respondents with good knowledge of folate-rich foods (AOR=0.3, 95% CI 0.1 to 0.7) and those who took iron and folic acid supplementation (AOR=0.6, 95% CI 0.4 to 0.9) during their pregnancy were less likely to develop folate deficiency.Conclusions In this study, a considerable proportion of pregnant women had folate deficiency during their pregnancy. Therefore, it is critical that nutritional treatment, education and counselling be strengthened to facilitate iron and folic acid supplementation during pregnancy.