Degeneration of retinal pigment epithelium cells (RPE) or delamination of RPE from Bruch’s membrane causes visual impairment. While regeneration of RPE cells is desirable to treat age-related macular degeneration (AMD) RPE cells of mammals, which are inherently non-renewable. Here, the bone marrow derived mesenchymal stem cells (BMSCs) that have the advantage of rapid growth and ease of extraction, which are adopted in order to help regenerate RPE cell. By co-culture with RPE cell, BMSCs exhibited a tendency to differentiate into RPE cell. By irradiating with the ultraviolet (UV) light, RPE cell was inhibited from growing and the differentiation of BMSCs was induced. The B-27® supplement was used as a differentiation factor, which helps BMSCs to differentiation into RPE cells. Specifically, B-27® supplement was mixed into the growth medium, which was added only to the co-culture and BMSCs groups. In consequence, UV-irradiated RPE cells did not grow, BMSCs in the co-culture group exhibited a tendency to differentiate into RPE cells. This was demonstrated by MTT assay, SEM, confocal imaging of the live/dead cells, RT-PCR and histological staining. Cell proliferation was the highest in the co-culture group, and the differentiation of BMSCs into RPE cells was observed in RT-PCR and histological staining. Our method will allow BMSCs with B-27® supplement to offer a new direction for RPE regeneration.