Toxin-antitoxin (TA) systems were initially discovered as plasmid addiction systems on low-copy-number plasmids through post-segregational killing. Thousands of TA loci have since been identified on chromosomes, plasmids and mobile elements in bacteria and archaea and have received increasing attention due to their diverse role in bacterial physiology and in maintenance of genetic elements. In this study, we identified and characterised a plasmid mediated type II TA system in Enterobacteriaceae. Protein sequence identity, secondary structure and conserved domain database search identified it as a member of the ParDE super family. This TA system is distributed in the IncI and IncF type plasmids of E. coli and other Enterobacteriaceae. Like the RK2 plasmid ParDE system, this novel systems (herein, ParDEI) inhibits bacterial growth and exhibits cell elongation upon toxin activation and functions as a plasmid addiction system in E. coli. Expression of this TA system increased 2-4 fold upon exposure to sub-minimum inhibitory concentration (MIC) doses of aminoglycoside, quinolone and beta-lactam antibiotics and ParDEI encodes tolerance of supra-MIC doses, with increased persister cell formation by ~100-1000 fold in E. coli. Additionally, this TA system provides a survival advantage in heat stress and the ParEI toxin promotes biofilm formation in E. coli. This novel plasmid addiction system increases persister cell and biofilm formation and promotes antibiotic and (heat) stress tolerance, and is likely to be an important factor in the success of IncI and IncF antibiotic resistance plasmids that carry it.