Folic acid (FA) food fortification paired with periconceptional FA supplementation leads to folate excess during pregnancy, which is increasingly associated with gestational diabetes mellitus (GDM) risk. Though the mechanisms remain unknown, we recently showed hormones that regulate insulin resistance and glucose homeostasis (placental growth hormone (GH2); placental lactogen (hPL)) are altered in pregnancies post-FA fortification. We hypothesized that FA acts directly on the placenta to promote trophoblast proliferation (placental growth) and/or function (hormone secretion). To address this hypothesis, early and mid-gestation placental villus explants (N=45; 6-16 weeks’ gestation) were treated with FA at 0 nM (acute deficiency), 10 nM (physiological deficiency), 40 nM (adequate), 200 nM (elevated) or 2000 nM (excess). Hormone secretion into culture media was measured 72 h post-treatment by ELISA. FA regulated GH2 and hPL in vitro. hPL secretion displayed a U-shaped response. Relative to the physiological norm (40 nM), 0 nM and 2000 nM FA increased hPL secretion by 40% (p = 0.009) and 25% (p = 0.08), respectively. GH2 concentration was also 25% higher in 2000 nM FA compared to 40 nM (p = 0.03). Real-time proliferation of placental cell lines revealed a similar biphasic relationship, where proliferation decreased with 0 nM and 2000 nM relative to 40 nM in JEG-3 and BeWo cytotrophoblasts, but not extravillous trophoblast HTR-8/SVneo. This research demonstrates FA affects placental endocrine function in vitro in early and mid-gestation human placentae. Interestingly, FA also affected proliferation of cells from syncytiotrophoblast lineage (hormone secreting trophoblasts) only. The effects of excess FA parallel those of acute deficiency, suggesting FA uptake and/or action is dysregulated in high-FA conditions, resulting in a deficient-like state. In the context of widespread FA fortification and supplementation in Australia, determining the effects of high FA intake on pregnancy health is essential.