In synaptic transmission, synaptic vesicles (SVs) are used at an active zone to release neurotransmitter into the synaptic cleft. SVs are finite and must be replenished rapidly. Previous work suggest that the multimodal scaffolding protein Intersectin-1 (ITSN1) is necessary for this process. ITSN1 interacts with multiple proteins at synapses, including Synapsin 1 and Endophilin A1. Synapsin 1 is localized on SVs and sequesters most SVs away from release sites. Synapsin 1 must be dispersed from vesicles by interactions with ITSN1 for SVs to be usable. Endophilin A1 is an endocytic protein that mediates SV recovery from the plasma membrane; its interaction with ITSN1 is necessary for recycling vesicles back to the active zone. Thus, ITSN1 seems to control the number of vesicles that can be readily used at synapses. However, the mechanism for vesicle replenishment is still unknown. To explore ITSN1’s role in synaptic vesicle replenishment, we overexpressed the wild-type form of ITSN1 and a mutant form containing a single point mutation that disrupts the interaction with Endophilin A1, ITSN1ΔEndoA1, in HEK-293 cells. Interestingly, when over-expressed alone, all forms of ITSN1 formed distinct liquid-like condensates. FRAP and 1,6-hexanediol drug treatments of transfected cells confirm the presence of distinct liquid condensates instead of protein aggregates. Liquid-like condensates also formed in double transfections of ITSN1ΔEndoA1 and Endophilin A1. However, this phenotype was not observed when the wild-type form of ITSN1 and Endophilin A1 were overexpressed together, indicating that Endophilin A1 disrupts ITSN1’s ability to phase separate in native conditions. Based on these results, we propose that ITSN1’s ability to recruit SVs for replenishment is linked to its ability to phase separate and for these phases to interact with Endophilin A1. Our findings pave the way for future investigations in ITSN1’s liquid-liquid phase separation as a mechanism for synaptic vesicle recruitment.