Previous studies have shown that TM9SF4 interacts with glycine-rich transmembrane domains (TMDs) and promotes their surface localization, presumably by escorting them along the secretory pathway. Our results indicate that TM9SF4 interacts with and sorts a variety of TMDs. In HEK cells, a TMD carrying a positively charged residue (T-R1) or a negatively charged residue (T-D1) was localized in the ER, but was partially relocated to the Golgi complex upon overexpression of TM9SF4. These results show that TM9SF4 controls the sorting of TMDs at the ER-Golgi interface. Remarkably, sorting of T-R1 in HCT116 cells was different from that in HEK cells: in HCT116 cells a substantial fraction of T-R1 was localized to the Golgi apparatus, and it was relocated to the ER by genetic ablation of TM9SF4. This observation indicates that TM9SF4 sorting activity differs in HEK and HCT116 cells, resulting in different sorting of TMDs in these two cell types. Although TM9SF1 associated with several TMDs, it did not visibly alter their intracellular transport in the secretory pathway and may function in other intracellular transport pathways.