Conjugative plasmids are the principal vector in the emergence and spread of antibiotic resistance genes in Enterobacteriaceae. Plasmid entry-exclusion (EEX) system can inhibit their transfer into the recipient bacteria carrying identical or very closely related plasmids and thought to be the common feature of conjugative plasmids. In this study, we have identified and characterised a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, responsible for the dissemination of blaKPC carbapenemase gene in Enterobacteriaceae. The identified eex gene in the recipient strain of different Enterobacteriaceae species inhibits the conjugation of pKPC_UVA01 plasmids at a range of 200-400 folds, and this inhibition was found to be a dose-dependent of the EEX protein in the recipient cells. The C-terminus truncated version of eex can alleviate the conjugative transfer inhibition effect. Unlike the strict specificity of plasmid exclusion by known EEX protein, the newly identified EEX in the recipient strain can inhibit the transfer of an IncP and an IncN plasmid. The deletion of eex gene from the plasmid pKPC_UVA01 does not interfere with its conjugative transfer but found to be essential in the donor bacteria for entry exclusion of this plasmid. To our knowledge, this is the first report of a single protein that is essential in both donor and recipient bacteria for entry exclusion of a plasmid. This eex gene is found to be distributed in the multi-drug resistance plasmids similar to pKPC_UVA01 in the different Enterobacteriaceae species and may contribute to the stability of this plasmid type by controlling its transfer.