Rhodopsin is a photoreceptive protein present in vertebrate rod photoreceptor cells, which are responsible for scotopic vision. Recent molecular studies have shown that several aquatic vertebrate species have independently acquired rhodopsin containing Asp83Asn, Glu122Gln, and Ala292Ser substitutions, causing a blue shift in the rhodopsin absorption spectra for adaptation to the blue-green photic environment in deep water. Here, we provide new evidence for the evolutionary and functional relevance of the Asp83Asn substitution. Spectroscopic and kinetic analyses of rhodopsins in six cichlid fishes from the East African Great Lakes using charge-coupled device spectrophotometer revealed that the Asp83Asn substitution accelerated the formation of meta-II, a rhodopsin intermediate crucial for activation of the G-protein transducin. Because rapid formation of meta-II likely results in effective transduction of photic signals, it is reasonable to assume that deep-water cichlid species have acquired rhodopsin containing Asn83 to adapt to dim lighting. Remarkably, rhodopsin containing Asn83 has been identified in terrestrial vertebrates such as bats, and these rhodopsin variants also exhibit accelerated meta-II formation. Our results indicated that the Asp83Asn substitution observed in a variety of animal species was acquired independently in many different lineages during vertebrate evolution for adaptation to dimly lit environments.