Syndapin-I is a synaptically enriched member of the F-BAR (FCH-BIN amphiphysin RVS) family of proteins. It consists of two functional domains; an N-terminal F-BAR, which can bind to and deform phospholipid membranes, and a C-terminal src homology 3 (SH3) flanked by a middle NPF-rich variable region. Syndapin-I is an important regulator of activity-dependent bulk endocytosis (ADBE) of synaptic vesicles (SV). It is an in vitro and in vivo phospho-protein in rat brains and nerve terminals. We performed proteomic analysis using the latest quantitative SWATH-MS technology to identify syndapin-I associated proteins and construct a syndapin-I interactome. Our analysis identified known and new proteins involved in intracellular trafficking, ion-transporters, and glycolytic and the Krebs cycle metabolic pathways, binding to the different syndapin-I functional domains and region. Protein domain and motif mapping of the interactors revealed that fructose 6-bisphosphate aldolase A (ALDOA), a glycolytic enzyme with moonlighting functions, binds the syndapin-I NPF-rich variable region. Mutational analysis of the amino acids in the variable region using protein-binding assays show that Trp (W)-357 residue in an acidic patch flanking the Ser-358 phosphosite (350-GQTYATEWSDDE-361) is the direct ALDOA binding site in syndapin-I. Therefore, ALDOA is a new in vitro syndapin-I binding protein. In non-neuronal cells, cytosolic syndapin-I changed to tubular-vesicular localisation when co-expressed with ALDOA, and the interaction is involved in receptor-mediated endocytosis. This suggests a role for the syndapin-ALDOA interaction in regulating endocytosis and supports the utility of the interactome as a screen for functionally important novel syndapin-I regulating proteins.