Endosomal acidification and transport are essential functions in signal transduction. Recent data suggest that Wnt signaling requires intact endosomal transport machinery but the effects of endosomal acidification on Wnt signal transduction have not been evaluated. Here we report that bafilomycin, a specific inhibitor of the vacuolar proton ATPase that blocks endosomal acidification, inhibits canonical Wnt signal transduction initiated by Wnt ligand and partially inhibits signaling initiated by disheveled. Bafilomycin does not affect Tcf promoter activation by beta-catenin. These data indicate that endosomal acidification is necessary for Wnt signaling. To identify interactions between endosomal transport proteins and Wnt receptors, we performed a GST fusion protein pulldown experiment and identified a possible indirect interaction between the LRP6 intracellular domain and vacuolar protein sorting protein 35 (VPS35). We show that an N-terminal deletion mutant of VPS35 reduces canonical Wnt signaling in HEK-293 cells expressing exogenous Wnt-1. These data suggest that endosomal V-type ATPase activity and retromer trafficking proteins are functionally important in Wnt signal transduction.