Cytosine　arabinoside　(Ara-C)　is　one　of　the　most　widely　used　chemotherapeutic　agents　for　hematological　malignancies.　The　residues　of　Ara-C　have　been　detected　in　wastewater　and　river　water　with　increased　usage　and　discharge.　As　the　ability　to　cross　the　placenta　and　the　teratogenicity　at　low　ng/L　levels,　the　toxic　effects　on　pregnant　women　and　infants　have　been　concerned.　The　toxicity　of　Ara-C　exposure　on　early　embryonic　neurodevelopment　has　not　been　fully　elucidated.　In　this　study,　pregnant　C57BL/6　mice　were　injected　with　different　doses　of　Ara-C　on　Gestation　day　(GD)　7.5　and　assessed　on　GD11.5　and　GD13.5　to　explore　the　neural　developmental　effects　of　Ara-C.　HE　staining,　immunofluorescence,　western　blot,　EdU　assay,　and　flow　cytometry　were　utilized　to　determine　the　toxic　effects　of　Ara-C　in　vivo　and　in　vitro.　Our　results　showed　that　Ara-C　(15–22.5　mg/kg　body　weight)　induced　the　occurrence　of　neural　tube　defects　(NTDs).　The　expression　of　PH3　was　markedly　reduced　in　embryos　with　Ara-C-induced　NTDs,　compared　to　the　control　group　(P　＜　0.05).　In　contrast,　cell　apoptosis　was　markedly　increased.　Increased　expression　levels　of　GFAP　and　decreased　Nestin　were　observed　in　the　embryonic　brain　tissues　in　Ara-C　induced　NTDs.　The　level　of　β-catenin　was　also　decreased　on　both　GD11.5　and　GD13.5.　These　results　were　confirmed　in　vitro　using　mouse　Sv129　embryonic　stem　cells　(mESC).　Ara-C　at　a　dose　comparable　to　the　environment　level　(0.05　nM)　had　cytotoxicity.　Impaired　Wnt/β-catenin　signaling　pathway　is　involved　in　Ara-C　exposure　induced　imbalance　between　cell　proliferation,　apoptosis,　and　differentiation,　which　might　contribute　to　Ara-C-induced　occurrence　of　NTDs.　Our　data　indicated　the　environmental　concentration　of　Ara-C　had　cytotoxicity　and　that　maternal　exposure　to　Ara-C　induced　NTDs.　These　results　might　provide　more　information　to　understand　the　environmental　toxic　impact　of　Ara-C　on　neurodevelopment.　©　2023　The　Authors