In summary, Otx2 could enhance the differentiation of MC‐RSCsinto photoreceptor‐like cells. Knockdown of Crx and Nrl or the activation of Wnt pathway could significantly inhibit the differentiationof MC‐RSCs into photoreceptor‐like cells in vitro. Subretinal injectionof MC‐RSCs overexpressing Otx2 could promote the formation ofneonatal photoreceptor‐like cells which form synaptic connectionwith adjacent cells, and partially rescue photoreceptor function inMNU‐treated photoreceptor degeneration rat model. Therefore, ourfindings provide a new avenue for photoreceptor cell regenerationto treat retinal degenerative diseases.