Type II Toxin-antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes with diverse critical cellular functions such as defense against phages, biofilm formation, persistence and virulence. GCN5-related N -acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. Here, we describe a group of GNAT-Xre TA modules that are widely distributed among Pseudomonas species and even certain Gram-positive bacteria. We investigate one of its members PacTA (encoded by PA3270/PA3269) from Pseudomonas aeruginosa, and demonstrate that the toxin PacT positively regulates the iron acquisition in P. aeruginosa. Notably, other than arresting translation via acetylating aminoacyl-tRNAs, PacT could directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit expression of downstream iron-acquisition-related genes. We further show that expression of the pacTA locus is up-regulated in response to iron starvation and the absence of PacT causes biofilm formation defect and attenuated pathogenesis. Overall, these findings reveal a novel regulation mechanism of GNAT toxin that controls genes involved in iron uptake process and contributes to the bacterial virulence.