Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors (TFs) that underlie mammalian ZGA in vivo remains elusive. Here, we showed that OBOX, a PRD-like homeobox domain TF family (OBOX1-8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a 2-4 cell arrest accompanied by impaired ZGA. Maternal and zygotic OBOX redundantly supported embryonic development as Obox KO defects could be rescued by restoring either of them. Chromatin binding analysis revealed Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated RNA Pol II “pre-configuration”, as Pol II relocated from the initial 1-cell binding targets to ZGA gene promoters and distal enhancers. The impaired Pol II pre-configuration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of 1-cell Pol II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mESCs. Hence, these data demonstrate that OBOX regulates murine ZGA and early embryogenesis.