Ion pumping microbial rhodopsins are photochemically active membrane proteins, converting light energy into ion-motive-force for ATP synthesis. Nonlabens dokdonensis rhodopsin 2 (NdR2), was recently identified as a light-driven Na+ pump. However, few functional studies on NdR2 have been conducted to elucidate its mechanism of ion transport. By reconstituting NdR2 into liposomes, we proved that NdR2 functions as a light-driven Na+/H+ pump. As Na+ concentration increased, the dominant H+ pump activity switched to the Na+ pump activity at neutral pH. The inversion of pH change by the addition of CCCP at low Na+ further suggested that the transport of Na+ and H+ should coexist in NdR2. By increasing H+ concentration, the affinity for Na+ lowered, which was indicated by an increase in KM from ~31mM at pH ~7.5, to ~74mM at pH ~6.5. These results demonstrated that Na+ transport competed with H+ transport in NdR2, which was confirmed by the dominant H+ pump activity at pH ~5.7. Kinetic experiments using pyranine uncovered a transient H+ uptake, followed by an H+ release at the millisecond time scale in both Na+ and K+ solutions. Therefore, these NdR2 results may provide functional and kinetic insights into the ion transport mechanism in light-driven Na+ pumps.,Copyright © 2017 Elsevier B.V. All rights reserved.