© 2017 The Author(s). Background: In thylakoid membrane, each monomer of the dimeric complex of cytochrome b 6 f is comprised of eight subunits that are both nucleus- and plastid-encoded. Proper cytochrome b 6 f complex integration into the thylakoid membrane requires numerous regulatory factors for coordinated transport, insertion and assembly of the subunits. Although, the chloroplast-encoded cytochrome b 6 f subunit IV (PetD) consists of three transmembrane helices, the signal and the mechanism of protein integration into the thylakoid membrane have not been identified. Results: Here, we demonstrate that the native PetD subunit cannot incorporate into the thylakoid membranes spontaneously, but that proper integration occurs through the post-translational signal recognition particle (SRP) pathway. Furthermore, we show that PetD insertion into thylakoid membrane involves the coordinated action of cpFTSY, cpSRP54 and ALB3 insertase. Conclusions: PetD subunit integration into the thylakoid membrane is a post-translational and an SRP-dependent process that requires the formation of the cpSRP-cpFtsY-ALB3-PetD complex. This data provides a new insight into the molecular mechanisms by which membrane proteins integration into the thylakoid membrane is accomplished and is not limited to PetD.