During development, fertilization triggers totipotency establishment, featured by zygotic genome activation/embryonic genome activation (ZGA/EGA). Mouse embryonic stem cells (mESCs) occasionally cycle through a two-cell (2C)-like status with activated expression of Dux and its targeted ZGA genes. Here, we demonstrate that deficiency of histone variant H3.3 dramatically stimulates expression of ZGA genes in mESCs. Our analysis revealed that H3.3 directly associates with Dux locus and inhibits Dux expression, therefore it is an important upstream regulator of Dux. Our finding is further supported by transcriptome change in early mouse embryos with H3.3 knockdown. We suggest that proper H3.3 level in early embryos is important to orchestrate ZGA activity for totipotency establishment. (C) 2019 Elsevier Inc. All rights reserved.