A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K(+) or Na(+) and whether the helical movements that result in the canonical E → C conformational change in other microbial rhodopsins are conserved in DeNaR. Visible absorption maxima of DeNaR in its unphotolyzed (dark) state show an 8 nm difference between Na(+) and K(+) in decyl maltopyranoside micelles, indicating an influence of the cations on the retinylidene photoactive site. In addition, electronic paramagnetic resonance (EPR) spectra of the dark states reveal repositioning of helices F and G when K(+) is replaced with Na(+). Furthermore, the conformational changes assessed by EPR spin-spin dipolar coupling show that the light-induced transmembrane helix movements are very similar to those found in bacteriorhodopsin but are altered by the presence of Na(+), resulting in a new feature, the clockwise rotation of helix F. The results establish the first observation of a cation switch controlling the conformations of a microbial rhodopsin and indicate specific interactions of Na(+) with the half-channels of DeNaR to open an appropriate path for ion translocation.