MARCH comprises a recently identified family of transmembrane RING-finger proteins which is implicated in diverse biological functions, such as immune regulation, protein quality control, and membrane trafficking. We previously identified MARCH-II, as a binding partner of syntaxin 6, which plays a role in endosomal protein transport. In this paper, we describe the cloning and characterization of MARCH-III which is the closest homolog of MARCH-II. It is broadly expressed at relatively high levels in spleen, colon, and lung. An immunofluorescence study of HeLa cells demonstrated that MARCH-III is present in peripheral vesicles partially colocalized with transferrin receptor. Overexpression of MARCH-III resulted in the redistribution of TGN46 and strong inhibition of transferrin uptake. Immunoprecipitation studies revealed that MARCH-III is associated with syntaxin 6 and MARCH-II. Mutational analyses revealed that the PDZ-binding motif and RING finger are essential for the subcellular localization of MARCH-III and the inhibitory effect on transferrin uptake. The location, associated molecules, and effects of overexpression suggest that MARCH-III is involved in the regulation of vesicular trafficking in endosomes.