Intersectin-2L (ITSN-2L) is a long isoform of ITSN family, which is a multimodule scaffolding protein functioning in membrane-associated molecular trafficking and signal transduction pathways. ITSN-2L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain, suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. But the role of C2 domain is obscure in this process. Here we report the crystal structure of human ITSN-2L C2 domain at 1.56Å resolution. The sequence and structural alignment of ITSN-2L C2 domain with other members of C2 domain protein family indicate its vital cellular roles in membrane trafficking, the generation of lipid-second messengers and activation of GTPases. Moreover, our data show the possible roles of ITSN-2L C2 domain in regulating the activity of Cdc42.