Defects in ankyrin underlie many hereditary disorders involving the mis-localization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We find in Madin-Darby canine kidney (MDCK) and in other cultured cells that the 25 residue ankyrin-binding sequence of 1-Na,K-ATPase facilitates the entry of 11-Na,K-ATPase into the secretory pathway, and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin-dependency on the ER-to-Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of 1-Na,K-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of wild-type 1-Na,K-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, while the trafficking of other proteins remain unaffected. Similar phenotypes are also generated by shRNA-mediated knockdown of ankyrin-R or the depletion of ankyrin in semi-permeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane, but also early in the secretory pathway to facilitate the intracellular trafficking of 1-Na,K-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER-retention or mis-localization.