Cadmium (Cd) can influence germ cell development, and epigenetic events may be involved. However, there is no study on whether Cd can influence germ cells differentiation into ovarian granulosa cells (GCs), and more insight into the molecular mechanism of the effect of Cd on germ cell development from mouse embryonic stem (ES) cells into ovarian granulosa cells and investigation of appropriate epigenetic factors are of great importance. In this study, mouse ES cell differentiation into GCs was established in an in vitro model. Subsequently, different Cd concentrations of 0, 0.1, 0.3, and 1 and then 3.0, and 10.0 μmol/L were cultured in this in vitro model. We demonstrated that Cd treatment can interrupt ES cell differentiation into GCs by morphology and ultrastructure observation. Four specific markers (octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Nanog homeobox (Nanog), and Anti-müllerian hormone type II receptor (Amhr2)) were significantly changed as measured by quantitative real-time-PCR or Western blot (p < 0.05). Cd also significantly changed the DNA methylation of GC sites on the CpG island of Nanog according to the sequential mass ARRAYR methylation method (p < 0.05). The MeRIP-qPCR method was used to detect the levels of N6-methyladenosine (m6A) methylation modification of long noncoding RNA (lncRNA) 1281 and indicated that they were decreased (p < 0.05). Microarray chip analysis, miRNA screening, and bioinformatics were used to further explore the roles of marker regulation-related miRNAs, and 27 miRNAs were putatively related to Cd-interrupted differentiation in ES cells. These data indicated that Cd can interrupt ES cell differentiation into GCs and affect germ cell development, and the underlying mechanism may involve epigenetic mechanisms. •Cadmium can disrupt mouse embryonic stem cell differentiation into ovarian GCs.•Cadmium inhibits ES cells differentiation by interfering with transcription factors.•Epigenetic mechanisms are involved in the toxic effects of cadmium on cells.