The ε subunit of FoF1-ATPase/synthase (FoF1) plays a crucial role in regulating FoF1activity. To understand the physiological significance of the ε subunit-mediated regulation of FoF1in Bacillus subtilis, we constructed and characterized a mutant harboring a deletion in the C-terminal regulatory domain of the ε subunit (ε∆C). Analyses using inverted membrane vesicles revealed that the ε∆C mutation decreased ATPase activity and the ATP-dependent H+-pumping activity of FoF1. To enhance the effects of ε∆C mutation, this mutation was introduced into a ∆rrn8 strain harboring only two of the 10 rrn (rRNA) operons (∆rrn8 ε∆C mutant strain). Interestingly, growth of the ∆rrn8 ε∆C mutant stalled at late-exponential phase. During the stalled growth phase, the membrane potential of the ∆rrn8 ε∆C mutant cells was significantly reduced, which led to a decrease in the cellular level of 70S ribosomes. The growth stalling was suppressed by adding glucose into the culture medium. Our findings suggest that the C-terminal region of the ε subunit is important for alleviating the temporal reduction in the membrane potential, by enhancing the ATP-dependent H+-pumping activity of FoF1.