Previous work proposed that elevated ubiquinone-8 (Q8) promotes the osmotolerance of Escherichia coli by enhancing membrane stability. Given that Q8 is a respiratory electron carrier, and that respiration is temporarily inhibited by osmotic stress, we hypothesized that increased levels of Q8 may contribute to E. coli osmotolerance by restoring aerobic respiration. In support of this hypothesis, Q8 deficiency impaired bacterial growth, osmotolerance, respiration, and the activities of the H+-symporters ProP, and LacY. Diphenylhexatriene fluorescence anisotropy measurements showed that Q8 decreased the fluidity of E. coli lipid liposomes. However, Q8 had no effect on the osmotic activation of ProP in proteoliposomes, a system in which the protonmotive force can be imposed without respiration. These results indicate that elevated Q8 confers osmoprotection by stimulating aerobic respiration rather than by affecting membrane physical properties. This work provides the first evidence that the regulation of Q8 synthesis can afford respiratory protection under osmotic stress. NSERC; Grant Number: 301-014400-401277