Extracellular vesicles (EVs) are particles released not only from blood cells but also from various organs. EVs, which are lipid bilayer vesicles, contain proteins, DNAs, and RNAs. The RNA and proteins within EVs display cell-specific characteristics. EVs derived from tumor cells are identified as biomarkers with diagnostic accuracy exceeding 90% for early cancer detection. Furthermore, EV RNA in serum has serves as a biomarker for toxicity. EVs have been found in various body fluids, including saliva, tears, urine, and amniotic fluid. In this study, we aimed to investigate the potential use of EV RNA in amniotic fluid as an indicator of developmental toxicity. Pregnant mice were exposed to valproic acid (VPA), a developmental toxicant, at concentrations of 0, 300, or 600 mg/kg/day on gestational days (GDs) 9–11. The study involved measuring VPA concentration in maternal plasma and fetuses on GD11, fetal weight on GD15 and 18, and assessing external morphological abnormalities on GDs11, 15 and 18. Additionally, EVs were collected from fetal amniotic fluid, and a comprehensive gene expression analysis of EV RNA was conducted on GD15. As a result, the concentration of VPA in the fetuses was not associated with the implantation location. Additionally, the VPA-treated group exhibited intrauterine growth retardation and teratogenic effects, including neural tube defects and digit malformations. EV RNA analysis identified differentially expressed EV small RNAs, both suppressed and induced, in the VPA-treated group compared with the control (vehicle, 0.5% Methylcellulose) group. These findings suggest that EV RNA in amniotic fluid serve as an indicator of developmental toxicity.