GEP 100 (p100) was identified as an ADP-ribosylation factor (ARF) guanine nucleotide-exchange protein (GEP) that partially colocalized with ARF6 in the cell periphery. p100 preferentially accelerated guanosine 5[γ-thio]triphosphate (GTPγS) binding by ARF6, which participates in protein trafficking near the plasma membrane, including receptor recycling, cell adhesion, and cell migration. Here we report that yeast two-hybrid screening of a human fetal brain cDNA library using p100 as bait revealed specific interaction with α-catenin, which is known as a regulator of adherens junctions and actin cytoskeleton remodeling. Interaction of p100 with α-catenin was confirmed by coimmunoprecipitation of the endogenous proteins from human HepG2 or CaSki cells, although colocalization was difficult to demonstrate microscopically. α-Catenin enhanced GTPγS binding by ARF6 in vitro in the presence of p100. Depletion of p100 by small interfering RNA (siRNA) treatment in HepG2 cells resulted in E-cadherin content 3-fold that in control cells and blocked hepatocyte growth factor-induced redistribution of E-cadherin, consistent with a known role of ARF6 in this process. F-actin was markedly decreased in normal rat kidney (NRK) cells overexpressing wild-type p100, but not its GEP-inactive mutants, also consistent with the conclusion that p100 has an important role in the activation of ARF6 for its functions in both E-cadherin recycling and actin remodeling.