Mandatory folic acid (FA) food fortification was introduced in Australia in September 2009 to prevent neural tube defects. However, FA food fortification, paired with periconceptional FA supplement recommendations, often results in folic acid intake exceeding the upper tolerable limit (~1000 μg/d) but the consequences are not well established. Increasingly, high maternal FA is associated with gestational diabetes mellitus (GDM) but the mechanism is unknown. Thus, we hypothesized that excess FA dysregulates hallmarks of placental cell function including proliferation, migration, invasion and secretion of placental hormones that orchestrate maternal insulin resistance and pancreatic beta cell expansion. To assess the effect of exogenous FA on placental function, BeWo cells and first trimester placental villus explants (6-12 weeks’ gestation) were treated with FA at concentrations of 10 nM (deficiency), 40 nM (adequate), 200 nM (elevated) or 2000 nM (highly elevated). Following treatment, we used Real-Time Cell Analyzer (RTCA) xCELLigence analysis to assess proliferation and migration of BeWo cells in response to FA across 72 h in culture. Secretion of placental hormones by both BeWo cells and placental explants was measured using specific ELISA analysis of culture media. Measured hormones were placental growth hormone (GHv), placental lactogen (hPL) and prolactin (PRL). Collectively, this research will provide the first evidence of in vitro effects of high dose FA on trophoblast function and hormone secretion in early human placenta. In the current landscape of widespread FA fortification and prenatal supplementation, characterising the effects of excess FA intake on maternal and fetal health via those on the placenta is essential to ensuring it does no harm. Furthermore, this has important implications in understanding the complex factors which contribute to the development of GDM.