Plasmid-encoded toxin-antitoxin (TA) systems are known for their role in plasmid maintenance via post-segregational killing. Here, we identified an inactive type II TA system, MtvT/MtvA (MtvTA), encoded on the conjugative plasmid pPAD8 from the clinical Pseudomonas aeruginosa strain PAD8. Despite its annotation as a toxin, MtvT exhibited no detectable toxicity in our assays. Interestingly, the deletion of the MtvTA significantly increased the transfer efficiency of pPAD8 from PAD8 to P. aeruginosa strain PAO1. Functional assays revealed that the MtvTA complex negatively regulates plasmid transfer by binding to the promoters of dot/icm system genes. In addition, pPAD8ΔmtvTA attenuated the pathogenicity of the host strain compared to pPAD8, highlighting a regulatory role for MtvTA in virulence. Mechanistically, the MtvTA complex positively regulates the type III and type VI secretion systems and pyocyanin biosynthesis by directly binding to the promoters of exsA and rsmY/rsmZ and indirectly influencing lasI expression, respectively. These findings provide new insights into the regulatory roles of an inactive plasmid-encoded TA system, expanding our understanding of the interplay between plasmids and their bacterial hosts.