Carboxyl-terminus of AQP2 (AQP2c) undergoes post-translational modifications, including phosphorylation and ubiquitination, for the regulation of aquaporin-2 (AQP2) translocation and protein abundance. We aimed to identify novel proteins interacting with AQP2c. Recombinant AQP2c protein was made in E. coli BL21 (DE3) by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct (IMCD) tubule suspensions interacted with rat AQP2c bound to Ni²⁺-resin were subjected to LC-MS/MS proteomic analysis. Potential interacting proteins were identified, including vacuolar protein sorting-associated protein 35 (Vps35). Co-immunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were partly co-localized at the intracellular vesicles in the collecting duct cells. The role of Vps35 in the dDAVP-induced AQP2 regulation was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under the siRNA-mediated Vps35 knockdown. dDAVP-induced AQP2 up-regulation was less prominent in the cells with Vps35 knockdown. Moreover, AQP2 protein abundance was decreased to a greater extent during the withdrawal period after dDAVP stimulation under the Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) treatment, but not by MG132 (a proteasome inhibitor). Immunocytochemistry demonstrated that internalized AQP2 was more associated with lysosomal marker (LAMP-1) in the primary cultured IMCD cells under the Vps35 knockdown. Taken together, Vps35 interacts with AQP2c and depletion of Vps35 is likely to be associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2 protein.