It remains unclear which factors—maternal or embryonic—are responsible for reprogramming the mammalian epigenome following fertilisation. Recently, overexpression of double homeobox (DUX) transcription factors—a family of proteins conserved among placental mammals—was found to induce a totipotent-like state in human and murine stem cells. In humans and mice, DUX transcription is activated in zygotes, suggesting it could be an early regulator of embryonic genome activation (EGA). Moreover, DUX4 has been reported to interact with histone acetyltransferases (HATs) in human cells, suggesting it could also be involved in epigenetic reprogramming. However, it is unclear whether DUX proteins are essential for development, as knockout studies in mice have found variable effects on EGA and blastocyst formation. Moreover, the function of DUX in embryos has yet to be established in any other mammal, despite recent reports that mice may not be the most appropriate model for human development. Here, we evaluate the function of DUX during the first divisions of the developing bovine embryo. Public annotations of DUX4 in cattle were largely based on predictions from the analogous human locus, so bovine DUX4 transcripts were manually reconstructed from published short-read RNA-seq data. The predicted bovine DUX4 protein resembled human DUX4 (E-value 2e-42), including homology at the HAT-binding domain. Using qPCR and immunofluorescence, we find that DUX4 is absent in the oocyte but present in zygotes by 22 h post-fertilisation (hpf), similar to what has been described in humans. Notably, this is well before minor EGA in both species, suggesting that DUX4 may be the first activated gene in the developing embryo. Conversely, the only other DUX family member present in the bovine genome, DUXA, is not appreciably expressed until the 8-cell stage (major EGA). To determine the function of DUX proteins in bovine development, we depleted zygotes for either DUX4 or DUXA through electroporation with siRNA at 10–14 hpf. The knock-down was verified by RT-qPCR and immunofluorescence. We find that DUX4 and DUXA depletion significantly reduced blastocyst formation from 44% (56/128) for controls to 2.5% (3/122) and 5.5% (7/126), respectively. Moreover, DUX4 depletion causes downregulation of EGA-associated genes, suggesting that DUX4 is a major regulator of EGA, as in the mouse. Considering the interaction of DUX4 with HATs in human cells, it is possible that DUX4 activates target gene expression through histone acetylation. Using CUT&Tag, we are currently investigating the effect of DUX4 depletion on genome-wide histone acetylation. Overall, these data indicate that zygotic expression of DUX is conserved among placental mammals, and that these factors serve as key regulators of EGA.