3-Nitropropionic acid (3-NP) is a naturally occurring mycotoxin produced by various fungi and plants. Despite reports on its toxicity, the potential impact of 3-NP exposure on reproductive health remains elusive. To this end, we conducted an in vitro study to investigate the toxic effects of 3-NP on the developmental processes of mouse embryos. Our results suggested that exposure to 50 μM 3-NP resulted in significant pre-implantation developmental arrest , with most embryos arrested at the 2-cell stage, indicating disruption of normal development. Further analysis indicated that 3-NP exposure altered embryonic gene expression, disrupted zygotic genome activation and maternal gene degradation, and inhibited maternal-zygote transition. Moreover, it impaired mitochondrial dysfunction, causing dysfunctional cellular energy metabolism and elevated intracellular oxidative stress, culminating in increased DNA damage. Additionally, 3-NP exposure caused aberrant epigenetic modifications, particularly the upregulation of histone methylation levels, including elevated H3K27me3 and H3K9me3, which are strongly related to gene expression silencing. In summary, this study elucidates the in vitro toxic effects of 3-NP on mouse embryo development and highlights its potential adverse effects on female reproductive health.