In polarized epithelial cells, vectorial protein trafficking is important for transporting specific membrane proteins to generate distinct apical and basolateral membrane protein compositions. The Exocyst is a conserved hetero-octameric protein complex, which regulates different aspects of protein trafficking, including tethering of the Golgi-derived vesicles to target membranes. Two of the Exocyst subunits, Sec5 and Exo84, competitively bind to the small GTPases, RalA and RalB, in a GTP-dependent manner. Although Ral GTPases have been proposed to mediate assembly of Exocyst holocomplexes, we hypothesize that they actually serve to allosterically regulate Exocyst functions by promoting association or disassociation of additional factors. Previous studies have shown that active RalA, but not RalB, accelerated basolateral exocytosis of E-cadherin. In contrast, knockdown of RalB, but not RalA, disrupts endocytosis of E-cadherin. However, mechanisms by which association of Ral GTPases with Sec5 and Exo84 regulate basolateral protein trafficking remain unclear. Here we investigate roles of Ral GTPases and the Exocyst in regulating basolateral protein trafficking using Madin Darby canine kidney (MDCK) cells and RNA interference (RNAi) technology. We show that RalA, but not RalB, is required for basolateral exocytosis of vesicular stomatitis virus glycoprotein (VSV-G) in the MDCK cells. We combined immunofluorescent labeling and surface biotinylation assays to demonstrate that RalA regulates VSV-G trafficking through the distinct interactions with Sec5 and Exo84. We also show that a Ral-uncoupled Sec5 mutant, but not a Ral-uncoupled Exo84 mutant, inhibits E-cadherin exocytosis. These results suggested that RalA and the Exocyst are required for basolateral exocytosis, and that RalA-Sec5 and RalA-Exo84 interactions play different roles during this process. Our study may provide new insights into mechanisms regulating protein trafficking in epithelial cells, and potentially lead to development of new therapeutic targets for the treatment of diseases in which exocytosis is impaired, such as Polycystic kidney disease and diabetes.