Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes that are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on theOpn4(melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have fiveOpn4genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history ofOpn4genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon, and gar) fishes. Our synteny analysis suggests that the intron-lessOpn4gene emerged by retroduplication after the branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-lessOpn4gene became abundantly used without substantial changes in the molecular properties of the Opn4 protein. This stepwise evolutionary model ofOpn4genes is quite similar to that ofrhodopsingenes in the Actinopterygii. The unique acquisition ofrhodopsinandOpn4retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments.