Opsins, a member of the G protein-coupled receptor (GPCR) family form photoreceptor proteins by binding the chromophore retinal to underlie photoreception of animals, such as vision [1]. Jumping spiders are highly vision-dependent animals and jump accurately to capture their prey. We previously reported that the measurement of distances to the targets, so-called depth perception, is achieved based on defocused images captured by the second deepest layer of the four-layered retina, where a green-sensitive rhodopsin (Rh1) is localized but green light is not focused [2, 3]. Here we investigated the significance of the spectral sensitivity of jumping spider Rh1 in the depth perception mechanism by analyzing absorption spectra of Rh1s of various jumping spider and “non-jumping” spider species. Mutational analysis of recombinant spider Rh1s with the aid of ancestral sequence inference uncovered the spectral tuning sites for the evolution of the absorption spectrum of jumping spider Rh1. In addition, since optical control of GPCR signaling using bistable animal opsins has recently been developing [4, 5], we also demonstrate the optogenetic potential of jumping spider Rh1, a Gq-coupled bistable opsin, in GPCR signaling-mediated physiological responses in mice.