Upon entry into stationary phase, bacteria dimerize 70S ribosomes into translationally inactive 100S particles by a process called ribosome hibernation. Previously, we reported that the hibernation-promoting factor (HPF) of Listeria monocytogenes is required for 100S particle formation and facilitates adaptation to a number of stresses. Here, we demonstrate that HPF is required for the high tolerance of stationary-phase cultures to aminoglycosides but not to beta-lactam or quinolone antibiotics. The sensitivity of a Δhpf mutant to gentamicin was suppressed by the bacteriostatic antibiotics chloramphenicol and rifampin, which inhibit translation and transcription, respectively. Disruption of the proton motive force by the ionophore carbonyl cyanide m-chlorophenylhydrazone or mutation of genes involved in respiration also suppressed the sensitivity of the Δhpf mutant. Accordingly, Δhpf mutants had aberrantly high levels of ATP and reducing equivalents during prolonged stationary phase. Analysis of bacterial uptake of fluorescently labeled gentamicin demonstrated that the Δhpf mutant harbored increased intracellular levels of the drug. Finally, deletion of the main ribosome hibernation factor of Escherichia coli, ribosome modulation factor (rmf), rendered these bacteria susceptible to gentamicin. Taken together, these data suggest that HPF-mediated ribosome hibernation results in repression of the metabolic activity that underlies aminoglycoside tolerance. HPF is conserved in nearly every bacterial pathogen, and the role of ribosome hibernation in antibiotic tolerance may have clinical implications.