Using a quantum mechanical/molecular mechanical approach, the absorption wavelength of the retinal Schiff base was calculated based on 13 microbial rhodopsin crystal structures. The results showed that the protein electrostatic environment decreases the absorption wavelength significantly in the cation-conducting rhodopsin but only slightly in the sensory rhodopsin. Among the microbial rhodopsins with different functions, the differences in the absorption wavelengths are caused by differences in the arrangement of the charged residues at the retinal Schiff base binding moiety, namely, one or two counterions at the three common positions. Among the microbial rhodopsins with similar functions, the differences in the polar residues at the retinal Schiff base binding site are responsible for the differences in the absorption wavelengths. Counterions contribute to an absorption wavelength shift of 50-120 nm, whereas polar groups contribute to a shift of up to similar to 10 nm. It seems likely that protein function is directly associated with the absorption wavelength in microbial rhodopsins.