Objective:To establish a murine model of neural tube defects (NTDs) by inhibition of thymidylate synthase (TS) via a specific inhibitor, raltitrexed (RTX). Based on the model, The role of DNA damage, cell apoptosis and proliferation in RTX-induced NTDs were investigated. Methods: The adult C57BL/6J pregnant of 7.5 d mice were randomly divided into six groups: one was for control and the other five were RTX-treated groups. NTDs were induced by intraperitoneally injection of various doses of RTX (5.0, 10.0, 11.5, 13.5, 15.0 mg/kg body weight) on gestational day 7.5. Control mice were injected with 0.9% NaCl at equal volume. Neural tube closure was examined by hematoxylin-eosin (H&E) staining on gestational day 11.5 and TS activity was measured by radioactive method following RTX treatment. Levels of thymidylate (dTMP) and uridylate (dUMP) were detected by high performance liquid chromatography (HPLC). Western-blotting was performed to analyze the expressions of replication protein A2 (RPA2), γ-H2AX and caspase-3 in NTDs embryos induced by RTX. Cell proliferation was analyzed by immunohistochemical method. Results: At 11.5 mg/kg bw, RTX induced the highest incidence of NTDs (30.56%). TS activity was significantly reduced by RTX treatment. Besides, levels of dUMP were increased associated with decreased dTMP levels after RTX treatment. Furthermore, phosphorylation of RPA2 and γ-H2AX were significantly increased in RTX-induced NTDs. The expression of caspase-3 was significantly increased and cell proliferation was significantly decreased. Conclusion: Murine model of NTDs was successfully established by inhibition of TS via RTX. DNA damage and imbalance between apoptosis and proliferation were important events caused by impairment of dTMP biosynthesis, which may be one of the key mechanisms underlying the development of NTDs.