Neural tube defects (NTDs) are severe congenital anomalies affecting 1–2 infants per 1000 births, and are influenced by genetic and environmental factors, with DNA hypomethylation and methylation cycle suppression being key causes. In our earlier investigation, decitabine (DCT) caused multiple NTDs in embryonic zebrafish, supporting this hypothesis. Recent research has emphasized the importance of myo-inositol (MI) in embryonic development and its efficacy in reducing the risk of neural tube defects, even in cases resistant to folate. We aimed to examine the effect of MI on DCT-induced NTDs in an embryonic zebrafish model. The embryos were exposed to 1 mM DCT alone, 50 µM MI with 1 mM DCT, 100 µM MI with 1 mM DCT, and a control group for comparison. The development, hatching, mortality rates, neural tube malformations, and neural tube patterning of developing embryos were monitored and recorded. Exposure to MI significantly reduced the incidence of NTDs in developing embryos. At concentrations of 50 µM and 100 µM, MI provided 35% and 30% protection against DCT-induced neural tube malformation, respectively. Multiple NTDs were significantly reduced in the MI groups, with 1 mM DCT causing 95% defects, 50 µM MI with 1 mM DCT causing 50%, and 100 µM MI with 1 mM DCT causing 55% defects. The DCT-induced hatching delay was also reversed by MI treatment. Alizarin red staining and histopathological observations supported these observations. In the context of neural tube development, the protective effects of MI against DCT-induced NTDs could be attributed to its potential role in epigenetic regulation, which may influence genetic expression. © 2025 Elsevier B.V., All rights reserved.