Toxin–antitoxin (TA) systems found in diverse bacteria play important roles in their adaptation to changing environments. The toxin of the Fic-1–AntF TA pair from Pseudomonas bijieensis strain 2P24 inhibits bacterial DNA replication by attacking the subunit B of DNA gyrase (GyrB) via AMPylation, while the antitoxin AntF blocks its enzymatic activity by forming a stable protein complex. Although many proteins, including bacterial toxins, have been found to catalyze AMPylation, few enzymes involved in reversing this modification have been described. In this study, we found that the Fic-1–AntF complex functions as a deAMPylase to reverse GyrB modification imposed by Fic-1. Structural and genetic analyses of the Fic-1–AntF complex revealed that Glu28 of AntF is critical for catalysis. Thus, AntF not only functions to inhibit the activity of Fic-1 but also cooperates with the toxin to return the modified substrate to its native form by de-modification. Our results reveal a novel regulatory mechanism for bacterial toxin, which sheds light on the evolution of such enzymes, particularly those of multiple subunits.