Background <p>Vaginal bleeding in early pregnancy is associated with adverse pregnancy outcomes. Excess folic acid ingestion has shown potential adverse effects on pregnancy outcomes. However, little is known about the fetal sex-specific influence of excess folic acid supplementation on vaginal bleeding. We aimed to determine whether increasing folic acid supplementation is associated with vaginal bleeding in early pregnancy with respect to fetal sex.</p> Methods <p>Two prospective cohorts of nulliparous pregnant women were accessed: prior to (SCOPE, <i>n</i> = 1164) and post (STOP, <i>n</i> = 1300) folic acid food fortification in Australia. Logistic regression was used to examine the relationship between folic acid supplementation and vaginal bleeding severity with fetal sex, as well as the relationship between vaginal bleeding and pregnancy outcomes, after adjusting for confounders.</p> Results <p>Reported spotting and mild vaginal bleeding was more common in STOP, compared with SCOPE. Women from the two cohorts who experienced spotting and mild vaginal bleeding in pregnancy were more likely to have supplemented with ≥ 800&#xa0;μg folic acid daily and had higher serum folate concentration, compared with no folic acid supplementation. Women who supplemented with ≥ 800&#xa0;μg folic acid per day, and with a male bearing pregnancy, had &gt; twofold greater odds [adjOR 2.14, (95% CI) 1.15–3.99, SCOPE and STOP] of vaginal bleeding in early pregnancy which was more likely to be more severe compared to those who did not supplement. This association was stronger (adjOR 2.85, 95%CI 1.23–6.57) in the STOP cohort. Furthermore, vaginal bleeding was associated with spontaneous preterm birth (sPTB) without premature rupture of membranes, placental abruption and small for gestational age (SGA) and lower gestational age at birth, the latter of which showed a male-fetus dependence, based on total cases in the two cohorts combined.</p> Conclusions <p>This study reveals a novel association between fetal sex and vaginal bleeding during early pregnancy, which is influenced by high folic acid intake. Our data suggest &lt; 800&#xa0;μg/day folic acid supplementation post food fortification may be safer, especially in male-bearing pregnancy, consistent with guidelines that recommend 400&#xa0;μg/day folic acid to prevent neural tube defects. Vaginal bleeding in early pregnancy needs more clinical attention as it may be an indicator for later pregnancy adverse outcomes.</p> Trial registration <p>SCOPE and STOP studies were registered with Australian New Zealand Clinical Trial Registry<b> (</b>ACTRN12607000551493; Registration date 26th October 2007 and ACTRN12614000985684; Registration date 12th September 2014, respectively).</p>