Lifelong visual impairment occurs from retinal diseases due to the inability of photoreceptor cells to regenerate in mammals. We demonstrated that endogenous Müller cells (MCs) in mice differentiate into photoreceptor-like cells by intravitreal injection of four small-molecule compounds: tumor growth factor-β inhibitor, bone morphogenetic protein inhibitor, glycogen synthase kinase 3 inhibitor, and γ-secretase inhibitor. In vitro, the messenger RNA of rhodopsin (Rho) in MCs increased 30-fold, and 25% of cultured MCs expressed Rho protein 7 days after stimulation with these compounds. In vivo, Rho-positive cells were regenerated on day 7 after the intravitreal injection of four compounds, accompanied with recovery of Rho-derived scotopic function. Lineage tracing in mice treated with N-methyl-N-nitrosourea, a disease model of photoreceptor degeneration, showed that the regenerated Rho-positive cells were originated from endogenous MCs. Finally, the regeneration of Rho-positive cells was also induced in the retina of rd10 mice, a model with similar genetic mutation as humans. Notably, the intravitreal injection significantly reduced cone cell death in rd10 mice. This treatment could be a new strategy in retinal regenerative medicine where mammalian endogenous MCs are reprogrammed into photoreceptor cells independent of transplantation or gene transfer.