Significance Rifampin phosphotransferases (RPH) belong to a recently identified antibiotic-resistance protein family that inactivates rifampin, the first-line drug against tuberculosis, by phosphorylation. By determining the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations, we reveal a toggle-switch mechanism of the LmRPH catalytic process in which the C-terminal His domain swings between the ATP-binding domain and the rifampin-binding domain to transfer phosphate from ATP to rifampin. These structures explain the low substrate selectivity of RPH for the rifamycins. The residues involved in rifampin phosphorylation are identified also. The structural mechanism revealed in this study will guide the development of a new generation of rifamycins.