Ingestion of food toxins such as aflatoxin B1(AFB1) during pregnancy may impair fetal neurodevelopment. However, animal model results may not be accurate due to the species’ differences, and testing on humans is ethically impermissible. Here, we developed an in vitro human maternal–fetal multicellular model composed of a human hepatic compartment, a bilayer placental barrier, and a human fetal central nervous system compartment using neural stem cells (NSCs) to investigate the effect of AFB1on fetal-side NSCs. AFB1passed through the HepG2 hepatocellular carcinoma cells to mimic the maternal metabolic effects. Importantly, even at the limited concentration (0.0641 ± 0.0046 μM) of AFB1,close to the national safety level standard of China (GB-2761-2011), the mixture of AFB1crossing the placental barrier induced NSC apoptosis. The level of reactive oxygen species in NSCs was significantly elevated and the cell membrane was damaged, causing the release of intracellular lactate dehydrogenase (p< 0.05). The comet experiment and γ-H2AX immunofluorescence assay showed that AFB1caused significant DNA damage to NSCs (p< 0.05). This study provided a new model for the toxicological evaluation of the effect of food mycotoxin exposure during pregnancy on fetal neurodevelopment.