Purpose To investigate the effect of astrocyte elevated gene-1 (AEG-1) overexpression on the biological behaviour of human retinoblastoma (RB) cells and its possible mechanism. Methods Three human RB cell lines (SO-RB50, Y79 and WERI-RB1) were infected with AEG-1-GFP recombinant lentiviral vectors to induce AEG-1 overexpression, while the cells infected with negative lentiviral vectors and cells without any intervention formed control groups. Results All three RB cell lines showed an overexpression of AEG-1 after lentivirus infection (p < 0.001 for all three cell lines). The survival rate of RB cells increased (all p < 0.001) in the AEG-1 overexpressed groups when compared with the control groups. There was a decrease in G0/G1 cell cycle phase arrest and an accumulation in G2/M cell cycle phase in all three RB cell lines (p < 0.001), with an induction in the S phase in WERI-RB1 cells. It was paralleled by a downregulation of p21 and p27 proteins and an upregulation of the Cdc2 protein. The apoptosis rate of RB cells declined (p < 0.001) when AEG-1 was overexpressed, in association with an upregulation of Bcl-2 protein and a downregulation of Bax protein and cleaved caspase-3 proteins. Conclusions A lentivirus-mediated AEG-1 overexpression in RB cells led in vitro to a growth promotion and an apoptosis inhibition of human RB cells, associated with an upregulation of the Bcl-2 protein, a downregulation of the Bax protein and of cleaved caspase-3 proteins, and with alterations of the cell cycle. AEG-1 may be involved in the development and progression of RB.