AbstractObjectiveWhile maternal folate deficiency has been linked to poor pregnancy outcomes such as neural tube defects, anaemia and low birth weight, the relationship between folate and preterm birth (PTB) in the context of the US post-folic acid fortification era is inconclusive. We sought to explore the relationship between maternal folate status and PTB and its subtypes, i.e. spontaneous and medically indicated PTB.DesignObservational study.SettingBoston Birth Cohort, a predominantly urban, low-income, race/ethnic minority population at a high risk for PTB.ParticipantsMother–infant dyads (n 7675) enrolled in the Boston Birth Cohort. A sub-sample (n 2313) of these dyads had maternal plasma folate samples collected 24–72 h after delivery.ResultsUnadjusted and adjusted logistic regressions revealed an inverse relationship between the frequency of multivitamin supplement intake and PTB. Compared with less frequent use, multivitamin supplement intake 3–5 times/week (adjusted OR (aOR) = 0·78; 95 % CI 0·64, 0·96) or >5 times/week (aOR = 0·77; 95 % CI 0·64, 0·93) throughout pregnancy was associated with reduced risk of PTB. Consistently, higher plasma folate levels (highest v. lowest quartile) were associated with lower risk of PTB (aOR = 0·74; 95 % CI 0·56, 0·97). The above associations were similar among spontaneous and medically indicated PTB.ConclusionsIf confirmed by future studies, our findings raise the possibility that optimizing maternal folate levels across pregnancy may help to reduce the risk of PTB among the most vulnerable US population in the post-folic acid fortification era.