Rab35 is a small G protein involved in various cellular events including clathrin-dependent endocytosis, phagocytosis, and autophagy. DENND1B, a DENN family member, acts as a guanine nucleotide exchange factor (GEF) for Rab35 to convert it to the GTP-bound active form from the GDP-bound inactive form. DENND1B contains the DENN domain which harbors GEF activity for Rab35 in the N-terminus, while the clathrin binding motif and adaptor protein-2-interaction motif are at the C-terminus. In this study, we investigated the intracellular localization of DENN1B in various cell types and found novel DENND1B-localized gathered line structures in BS-C-1 cells and in some other cell types. The localization of DENND1B to gathered line structures was dependent on a specific region located in the C-terminus of DENND1B protein. DENND1B-localized gathered lines were partially associated with microtubules but not with F-actin; instead, F-actin bundles surrounded the assembly of gathered lines. We also show that the gathered line structures appeared at the bottom of spreading lamellipodia and disappeared at the retracting site during cell motility in EGF-stimulated BS-C-1 cells. These results shed light on a new role for DENND1B in the regulation of cell migration.