Renal tubular reabsorption is important for extra-cellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterised by low-molecular-weight-proteinuria, hypercalciuria, nephrolithiasis and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here we report that CLC-5 interacts with Kinesin Family Member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, Glutathione-S-Transferase (GST) pull-down and co-immunoprecipitation assays, the C-terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous co-immunoprecipitation of CLC-5 and KIF3B and co-distribution with endosomal markers in mouse kidney fractions. Confocal live-cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5 containing vesicles along KIF3B microtubules. KIF3B over-expression and under-expression, using siRNA, had reciprocal effects on whole-cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarised cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression, and for facilitating endocytosis and microtubular transport in the kidney. Key words: Chloride/proton antiporter, Kinesin family, proximal tubular reabsorption, hypercalciuric nephrolithiasis.