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The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is an essential bacterial respiratory enzyme that generates a Na+ gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na+ translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na+ translocation using a series of synthetic UQs with Vibrio cholerae Na+-NQR reconstituted into liposomes. UQ 0 that has no side chain and UQ CH3 and UQ C2H5 , which have methyl and ethyl side chains, respectively, were catalytically reduced by Na+-NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na+ translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na+ translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n -propyl (C 3 H 7) or longer significantly restored Na+ translocation. It has been considered that Na+ translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na+ translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na+ translocation. • Na+-NQR is a unique redox-driven Na+ pump found only in prokaryotes. • The mechanism responsible for the redox-driven Na+ translocation remains unknown. • The reduction of UQ 0 , which has no alkyl side chain, generated no membrane potential. • Na+ translocation was recovered by lengthening the chain to n -propyl (C3) or longer. • The coupling of UQ reduction to Na+ pumping critically depends on the UQ side chain. [ABSTRACT FROM AUTHOR]