Toll-like receptors (TLRs) are type I transmembrane (TM) proteins indispensable for sensing microbial and viral infection. Despite their conserved primary structures, some TLRs that detect pathogen-derived nucleic acids (TLR3, TLR7, TLR8, and TLR9) are retained in the cytoplasm. The intracellular localization of TLR9 is important for its ability to discriminate self- and non-self DNA, but the mechanism by which it is retained in the cytoplasm is unclear. In the present study, we found that the TM domain of TLR9 directs its intracellular localization. The TM domain of TLR9 also targets CD25, a heterologous type I TM protein, to intracellular compartments that contain TLR9. We also found that TLR9 generally co-localizes with TLR3, although its linker region, not its TM domain, directs intracellular localization of TLR3. These data demonstrate that the TM domain of TLR9 is a critical regulatory element that targets TLR9 to its intracellular location.