Microbial rhodopsins are distributed through many microorganisms. Heliorhodopsins are newly discovered but have an unclear function. They have seven transmembrane helices similar to type-I and type-II rhodopsins, but they are different in that the N-terminal region of heliorhodopsin is cytoplasmic. We chose 13 representative heliorhodopsins from various microorganisms, expressed and purified with an N-terminal His tag, and measured the absorption spectra. The 13 natural variants had an absorption maximum (λmax) in the range 530-556 nm similar to proteorhodopsin (λmax = 490-525 nm). We selected several candidate residues that influence rhodopsin color-tuning based on sequence alignment and constructed mutants via site-directed mutagenesis to confirm the spectral changes. We found two important residues located near retinal chromophore that influence λmax. We also predict the 3D structure via homology-modeling of Thermoplasmatales heliorhodopsin. The results indicate that the color-tuning mechanism of type-I rhodopsin can be applied to understand the color-tuning of heliorhodopsin.