In order to explore the effect of red light on the formation of skin pigment cells, CRISPR/Cas9 gene editing technology was used to construct a zebrafish line with homozygous deletion of the red light opsin 2 gene opn1lw2. A red LED lamp with a light intensity of 800 ± 100 lx was used to irradiate 15-day-old AB strain wild-type zebrafish (Danio rerio) and opn1lw2-/- strain zebrafish for 60 days, the number of melanocytes in the dorsum skin of wild-type and mutant zebrafish were counted with Photoshop CS5，and the data were analysed using GrahPad Prsim6. The results showed that the number of melanocytes in the dorsum skin of wild-type zebrafish was significantly more than that of the mutant (Fig. 2a﹣c). Real-Time quantitative PCR (RT-PCR) analysis found that the expression of the melanocyte marker gene kit in the dorsum skin of wild-type zebrafish was significantly higher than that of the mutant (Fig. 2d). There was no significant difference in the expression of the xanthophore marker gene csf1ra and the iridophores marker gene pnp4a of the dorsum skin between wild zebrafish and the opn1lw2-/- mutant (Fig. 3c, d), indicating that red light can regulate the formation of melanocytes in the dorsum skin of zebrafish through the opn1lw2, but does not affect the formation of skin xanthophores and iridophores; moreover, it regulates the differentiation of melanocytes. The expression of the precursor gene pomca of α-MSH in the dorsum skin of opn1lw2-/- zebrafish was significantly lower than that of the wild type (Fig. 2e), indicating that red light regulates the expression of pomca gene through opn1lw2 to induce the formation of melanocyte; RT-PCR detection found that the expression of retinal dehydrogenase raldh3 in wild-type zebrafish skin was significantly higher than that of mutant opn1lw2-/- (Fig. 4b), while there was no difference in expression of raldh2 encoding retinal dehydrogenase (Fig. 4a), indicating that opn1lw2 may selectively mediate the red light-induced synthesis of retinoic acid to regulate the formation of melanocytes. These results are importantfor understanding the formation of pigment cells in fish skin induced by red light irradiation.