Staphylococcus aureus is an important pathogen that colonizes 20-50% of the human population. They often acquired antibiotic resistance genes situated on mobile genetic elements, such as plasmids. RNA decay is very important for gene expression regulation in bacteria where RNases are crucial players in RNA decay. We worked with a clinical strain SA564 harbouring a natural plasmid, pSA564. We have observed that the deletion of the gene encoding RNase J1, a 5' to 3' exoribonuclease and as endonuclease, leads to the loss of the otherwise stable pSA564 plasmid. The pSA564 plasmid is part of RepA_N family of replicons that encodes an essential plasmid-encoded replication initiator protein, RepA. We have shown that its expression is negatively regulated by an antisense RNA, RNA1. Here we have demonstrated that RNA1, especially some intermediates, are highly accumulated in ΔJ1 strain and our data strongly suggests that this is the reason for plasmid loss in a ΔJ1 strain. It is the first time that an RNase is implicated in plasmid replication control of staphylococcal multi-resistance plasmids therefore it might represent a reliable biological target for development of new drugs in the battle against bacterial drug resistance in S. aureus.