Objective To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium )or 10%FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin(13.33%)in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1(30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types. Methods To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM / F12 + N2 with EGF and bFGF (suspension medium) or 10% The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated as with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin (13.33%) in suspension medium; however, Nestin 60.27%, Pax6 52.2%, Map2 34.94% (38.17%), Thy- 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high (30.84%) and Rhodopsin (34.67% degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.