Packaging of DNA into nucleosomes not only helps to store genetic information but also creates diverse means for regulating DNA-templated processes. Attempts to reveal additional functions of the nucleosome have been unsuccessful, owing to cell lethality caused by nucleosome deletion. Taking advantage of the mammalian fertilization process, in which sperm DNA assembles into nucleosomes de novo, we generated nucleosome-depleted (ND) paternal pronuclei by depleting maternal histone H3.3 or its chaperone HIRA in mouse zygotes. We found that the ND pronucleus forms a nuclear envelope devoid of nuclear pore complexes (NPCs). Loss of NPCs is accompanied by defective localization of ELYS, a nucleoporin essential for NPC assembly, to the nuclear rim. Interestingly, tethering ELYS to the nuclear rim of the ND nucleus rescues NPC assembly. Our study thus demonstrates that nucleosome assembly is a prerequisite for NPC assembly during paternal pronuclear formation.