Abstract In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic regions. Regulated transcription from selected genes is established during the two-cell stage. This dramatic change in the gene expression pattern marks the initiation of the gene expression program and is essential for early development. We investigated the involvement of the histone variants H3.1/3.2 in the regulation of changes in gene expression pattern during the two-cell stage. Immunocytochemistry analysis showed low nuclear deposition of H3.1/3.2 in the one-cell stage, followed by a rapid increase in the late two-cell stage. Where chromatin structure is normally closed between the one- and two-cell stages, it remained open until the late two-cell stage when H3.1/3.2 were knocked down by small interfering RNA. Hi-C analysis showed that the formation of the topologically associating domain was disrupted in H3.1/3.2 knockdown (KD) embryos. Promiscuous transcription was also maintained in the late two-cell stage in H3.1/3.2 KD embryos. These results demonstrate that H3.1/3.2 are involved in the initial process of the gene expression program after fertilization, through the formation of a closed chromatin structure to execute regulated gene expression during the two-cell stage.