Actinorhodopsins are encoded by a distinct group of microbial rhodopsin (MR) genes predominant in non-marine actinobacteria. Despite their role in the global energy cycle and potential for bionanotechnological applications, our understanding of actinorhodopsin proteins is limited. Here, we characterized the actinorhodopsin RlActR from the freshwater actinobacterium Rhodoluna lacicola, which conserves amino acid residues critical for light-driven proton pumping found in MRs. RlActR was efficiently overexpressed in Escherichia coli in milligram amounts and isolated with high purity and homogeneity. The purified RlActR absorbed green light and its primary proton acceptor exhibited a mildly acidic apparent pK