Retinoblastoma is a childhood ocular tumor caused by the inactivation of both alleles of the retinoblastoma gene (Rb1). Without Rb1 gene function, chromosomal aberrations are observed in retinoblastoma cells. The instability of the genome is closely associated with the repair of DNA double-strand breaks (DSBs). However, the precise molecular mechanism of action of Rb1 in DNA DSB repair remains unclear. Thus, in this study, we aimed to investigate whether the Rb1 gene affects DNA stability by assaying DNA DSB repair and also whether it regulates the proliferation of retinoblastoma cells. Rb1 immunofluorescence and RT-PCR were performed, demonstrating that the Rb1 gene is silenced in SO-Rb50 retinoblastoma cells, and the karyotype analysis of SO-Rb50 cells indicated that the loss of Rb1 function led to genomic instability; both numerical and structural chromosomal aberrations were observed in our study. In addition, the DNA DSB repair efficiency of the SO-Rb50 cells was measured by γ-H2AX immunofluorescence, a commonly used in situ marker of DNA DSBs, following exposure to ionizing radiation (IR) (2.5 and 5.0 Gy). We found that the DNA repair efficiency was significantly increased following IR-induced damage (P<0.01). However, there was no significant difference in DNA repair efficiency between the cells expressing exogenous Rb1 and the control (P>0.05). The assay for the screening of the effect of Rb1 on the sub-pathway of DNA DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated that Rb1 did not affect NHEJ activity, although it significantly promoted the HR pathway (HR levels increased by 2.46-fold) compared with the control (P<0.01). Furthermore, we found that the cell viability of the SO-Rb50 cells transfected with exogenous Rb1 was significantly inhibited (P<0.01) and cell cycle assay indicated that exogenous Rb1 induced S phase arrest (P<0.001) which also inhibited the proliferation of retinoblastoma cells (SO-Rb50) in vitro. Therefore, this study provides new insight into the mechanisms of action of the Rb1 gene in regulating the proliferation of retinoblastoma cells.