Objective(s): Non-invasive treatment options for retinoblastoma (RB), the most common malignant eye tumor among children, are lacking. Epithelial growth factor receptor (EGFR) accelerates cell proliferation, survival, and invasion of many tumors including RB. However, RB treatment by targeting EGFR has not yet been researched. In the current study, we investigated the effect of EGFR down-regulation on RB progression using shRNA lentiviral vectors. Materials and Methods: EGFR expression in Weri-Rb-1 cells was down-regulated by EGFR shRNA-bearing lentiviral vectors. Cell death, proliferation, cell cycle as well as invasion after EGFR down-regulation were determined. Further signaling pathway analysis was done by Western blot. Results: Our results revealed that EGFR shRNA could specifically down-regulate EGFR expression and down-regulation of this protein promoted cell death. Further analysis on cell cycle demonstrated that EGFR down-regulation also suppressed cell proliferation by arresting cells at G1 phase. Invasion analysis showed that EGFR down-regulation suppressed cell invasion and was correlated with alteration in the expression of matrix metalloproteinases 2 and 9. Further signaling pathway analysis revealed that EGFR down-regulation mediated RB progression was through PI3K/AKT/mTOR signaling pathway. Conclusion: Our study revealed that EGFR down-regulation, through the PI3K/AKT/mTOR signaling pathway, could inhibit RB progression by promoting cell death while suppressing cell proliferation and invasion. The findings of our study indicated that down-regulation of EGFR using shRNA lentiviral vectors may offer a novel non-invasive treatment for RB.