Photoactive proteins with cognate chromophores are widespread in organisms, and function as light-energy converters or receptors for light-signal transduction. Rhodopsins, which have retinal (vitamin A aldehyde) as their chromophore within their seven transmembrane α-helices, are classified into two groups, microbial (type-1) and animal (type-2) rhodopsins. In general, light absorption by type-1 or type-2 rhodopsins triggers a trans-cis or cis-trans isomerization of the retinal, respectively, initiating their photochemical reactions. Recently, we found a new microbial rhodopsin (middle rhodopsin, MR), binding three types of retinal isomers in its original state: all-trans, 13-cis, and 11-cis. Here, we identified the absolute absorption spectra of MR by a combination of high performance liquid chromatography (HPLC) and UV-vis spectroscopy under varying light conditions. The absorption maxima of MR with all-trans, 13-cis, or 11-cis retinal are located at 485, 479, and 495 nm, respectively. Their photocycles were analyzed by time-resolved laser spectroscopy using various laser wavelengths. In conclusion, we propose that the photocycles of MR are MR(trans) → MR(K):lifetime = 93 μs → MR(M):lifetime = 12 ms → MR, MR(13-cis) → MR(O-like):lifetime = 5.1 ms → MR, and MR(11-cis) → MR(K-like):lifetime = 8.2 μs → MR, respectively. Thus, we demonstrate that a single photoactive protein drives three independent photochemical reactions.