Interferon-inducible transmembrane proteins (IFITM1/2/3) have been reported to suppress the entry of a wide range of viruses. However, their antiviral functional residues and specific mechanisms are still unclear. Here, we firstly resolved the topology of IFITM1 on the plasma membrane where N-terminus points into the cytoplasm and C-terminus resides extracellularly. Further, KRRK basic residues of IFITM1 locating at 62-67 of the conserved intracellular loop (CIL) were found to play a key role in the restriction on the Zika virus (ZIKV) and dengue virus (DENV). Similarly, KRRK basic residues of IFITM2/3 also contributed to suppressing ZIKV replication. Finally, IFITM1 was revealed to be capable of restricting the release of ZIKV particles from endosome to cytosol so as to impede the entry of ZIKV into host cells, which was tightly related with the inhibition of IFITM1 on the acidification of organelles. Overall, our study provided topology, antiviral functional residues and the mechanism of interferon-inducible transmembrane proteins.