To observe the effect of low oxygen concentration on the neural retina in human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs).The hiPSC and a three-dimensional culture method were used for the experiments. Generated embryoid bodies (EBs) were randomly and equally divided into hypoxic and normoxic groups. Photographs of the EBs were taken on days 38, 45, and 52, and the corresponding volume of EBs was calculated. Simultaneously, samples were collected at these three timepoints, followed by fixation, sectioning, and immunofluorescence.The proportion of Ki67-positive proliferating cells increased steadily on day 38; this proliferation-promoting effect tended to increase tissue density rather than tissue volume. On days 45 and 52, the two groups had relatively similar ratios of Ki67-positive cells. Further immunofluorescence analysis showed that the ratio of SOX2-positive cells significantly increased within the neural retina on day 52 (P<0.05). In contrast, the percentage of PAX6- and CHX10-positive cells significantly decreased following hypoxia treatment at all three timepoints (P<0.01), except for CHX10 at day 45 (P>0.05). Moreover, the proportion of PAX6-/TUJ1+ cells within the neural retinas increased considerably (P<0.01, <0.05, <0.05 respectively).Low oxygen promotes stemness and proliferation of neural retinas, suggesting that hypoxic conditions can enlarge the retinal progenitor cell pool in hiPSC-derived ROs.International Journal of Ophthalmology Press.