The nuclear envelope (NE) needs to be reassembled after each mitotic cycle and also after fertilization, it is important to understand the regulation and dynamics of this universally conserved membrane fusion phenomenon. In this work we report the requirement of Class I PI3-Kinases for the NE formation in vitro, and its involvement in the fusion of male and female pronuclear membranes. We show for the first time that PtdIns(3,4,5)P₃ in vivo is located in the NE precursor membrane vesicles around the male pronucleus and at the vicinity of both male and female pronuclei prior to the fusion of membranes. Using coincidence amplified-FRET monitored by fluorescence lifetime imaging microscopy (FLIM) we determine the lipid-protein interaction between PtdIns(3,4,5)P₃ and the small GTPase Rab7 during pronuclear membrane fusion leading to zygote NE formation. Our work implies a direct role of polyphosphoinositides and their modifying enzymes in the NE assembly and the early stages of embryo formation.