In contrast to the case in mammals, the fish optic nerve can spontaneously regenerate and visual function can be fully restored 3–4 months after optic nerve injury (ONI). However, the regenerative mechanism behind this has remained unknown. This long process is reminiscent of the normal development of the visual system from immature neural cells to mature neurons. Here, we focused on the expression of three Yamanaka factors (Oct4, Sox2, and Klf4: OSK), which are well-known inducers of induced pluripotent stem (iPS) cells in the zebrafish retina after ONI. mRNA expression of OSK was rapidly induced in the retinal ganglion cells (RGCs) 1–3 h after ONI. Heat shock factor 1 (HSF1) mRNA was most rapidly induced in the RGCs at 0.5 h. The activation of OSK mRNA was completely suppressed by the intraocular injection of HSF1 morpholino prior to ONI. Furthermore, the chromatin immunoprecipitation assay showed the enrichment of OSK genomic DNA bound to HSF1. The present study clearly showed that the rapid activation of Yamanaka factors in the zebrafish retina was regulated by HSF1, and this sequential activation of HSF1 and OSK might provide a key to unlocking the regenerative mechanism of injured RGCs in fish.