We clarified the properties of visual opsin genes in the marbled sole (Pseudopleuronectes yokohamae) by cDNA sequencing, quantification of the opsin gene expression from the larval to the juvenile stage, and measurement of the maximum absorption spectra (1max) using photopigment reconstitution. In the marbled sole eye, at least eight visual opsin genes, lws, rh2-a, rh2-b, rh2-c, sws2a, sws2b, sws1, and rh1, were expressed. Quantitative RT-PCR analysis revealed that the expression of opsin genes increased (lws, rh2-c, sws2a, and rh1) or decreased (rh2-a, rh2-b, sws2b, and sws1) from the larval to the juvenile stage. Notably, rh2-a expression was observed only in preto mid-metamorphic stage larvae and disappeared after metamorphosis. Thus, pre-metamorphism-specific expression of rh2-a in the marbled sole suggests that its function is restricted to the developmental stage. The reconstituted RH2-A opsin 1max was 470 nm, which is typical of acanthopterygian species. These results strongly suggest that mid-wavelength-sensitive rh2-a expression was diminished drastically in the marbled sole, probably resulting in a shift of spectral sensitivity during its metamorphosis from the larval to the juvenile stage.