Bacterial conjugation is the process by which plasmids are mobilised from one bacterium (donor) to another (recipient) in a contact-dependent manner. It is essential for the transfer of antibiotic resistant plasmids in clinically pervasive pathogens. Conjugative transfer depends on a type IV secretion system (T4SS) which can be classified according to homology between T4SS components: MPFF, MPFT, MPFI and MPFG. The conjugation efficiency of F-like plasmids, which utilise a MPFF T4SS, depends on the formation of a mating pair that is mediated by mating pair stabilisation (MPS). After plasmid transfer, transconjugants are prevented from acquiring a second copy of the same plasmid or a closely related plasmid, which is mediated by surface exclusion (SFX) and entry exclusion (EEX) mechanisms. In this study the molecular and structural basis of MPS and SFX were investigated in the context of F-like conjugation. It was shown that MPS is mediated by intimate interaction between a plasmid encoded protein TraN on the donor cell with a unique outer membrane (OM) protein on the recipient cell. Specifically, TraN from the F-like plasmid pKpQIL (TraNpKpQIL) forms a complex with the OM porins OmpK35 and OmpK36, while TraN from the F-like plasmid R100-1 (TraNR100-1) forms a complex with the OM porin OmpW. Moreover, the structure and function of the SFX protein TraT was studied. The cryo-EM structures of TraT from pKpQIL (TraTpKpQIL) and the F plasmid (TraTF) were solved to 2.47 Å and 2.66 Å respectively. The sites of TraTpKpQIL conferring pKpQIL SFX specificity were probed through the generation of TraT chimeras. Overall, these findings contribute to a greater understanding of MPS and SFX, two key processes of F-like plasmid conjugation. These data could potentially inform strategies to reduce the spread of antibiotic resistance genes.