Rhodopsin is a large family of retinal-binding photoreceptive proteins found in animals and microbes. The retinal chromophore is normally positively charged by protonation of the Schiff base linkage, which is stabilized by the negatively charged counterion(s) such as aspartates, glutamates, and chloride ions. In contrast, no cation binding was reported near the retinal chromophore under physiological pH, presumably because of the electrostatic repulsion. Sodium binding takes place in light-driven sodium pumps, but the binding near the retinal chromophore is a transient event. Here, we report Ca2+ binding to a wild-type microbial rhodopsin, which is achieved for the neutral retinal chromophore with a deprotonated Schiff base. TAT rhodopsin from marine bacteria contains protonated and deprotonated retinal Schiff bases at physiological pH (pH ∼ 8), which absorb visible and UV light, respectively. We observed that the equilibrium shifted toward the deprotonated state upon increasing Ca2+ concentration, and the Kd value was determined to be 0.17 mM. Site-directed mutagenesis study showed that E54 and D227 constitute the binding site of Ca2+. ATR-FTIR spectroscopy revealed secondary structural changes upon Ca2+ binding to E54 and D227, while they are negatively charged with or without Ca2+ binding.