ImportancePrenatal perfluoroalkyl and polyfluoroalkyl substances (PFAS) have been linked to adverse birth outcomes. Previous research showed that higher folate concentrations are associated with lower blood PFAS concentrations in adolescents and adults. Further studies are needed to explore whether prenatal folate status mitigates PFAS-related adverse birth outcomes.ObjectiveTo examine whether prenatal folate status modifies the negative associations between pregnancy PFAS concentrations, birth weight, and gestational age previously observed in a US cohort.Design, Setting, and ParticipantsIn a prospective design, a prebirth cohort of mothers or pregnant women was recruited between April 1999 and November 2002, in Project Viva, a study conducted in eastern Massachusetts. Statistical analyses were performed from May 24 and October 25, 2022.ExposurePlasma concentrations of 6 PFAS compounds were measured in early pregnancy (median gestational week, 9.6). Folate status was assessed through a food frequency questionnaire and measured in plasma samples collected in early pregnancy.Main Outcomes and MeasuresBirth weight and gestational age, abstracted from delivery records; birth weight z score, standardized by gestational age and infant sex; low birth weight, defined as birth weight less than 2500 g; and preterm birth, defined as birth at less than 37 completed gestational weeks.ResultsThe cohort included a total of 1400 mother-singleton pairs. The mean (SD) age of the mothers was 32.21 (4.89) years. Most of the mothers were White (73.2%) and had a college degree or higher (69.1%). Early pregnancy plasma perfluorooctanoic acid concentration was associated with lower birth weight and birth weight z score only among mothers whose dietary folate intake (birth weight: β, −89.13 g; 95% CI, −166.84 to −11.42 g; birth weight z score: −0.13; 95% CI, −0.26 to −0.003) or plasma folate concentration (birth weight: −87.03 g; 95% CI, −180.11 to 6.05 g; birth weight z score: −0.14; 95% CI, −0.30 to 0.02) were below the 25th percentile (dietary: 660 μg/d, plasma: 14 ng/mL). No associations were found among mothers in the higher folate level groups, although the tests for heterogeneity did not reject the null. Associations between plasma perfluorooctane sulfonic acid and perfluorononanoate (PFNA) concentrations and lower birth weight, and between PFNA and earlier gestational age were noted only among mothers whose prenatal dietary folate intake or plasma folate concentration was in the lowest quartile range. No associations were found among mothers in higher folate status quartile groups.Conclusions and RelevanceIn this large, US prebirth cohort, early pregnancy exposure to select PFAS compounds was associated with adverse birth outcomes only among mothers below the 25th percentile of prenatal dietary or plasma folate levels.