Purpose: The purpose of this study was to investigate the pro-apoptotic effect of camptothecin (CPT) on Y79 retinoblastoma cells and the role of Forkhead box, class O (FOXO1) in CPT-induced apoptosis. Methods: CPT-induced apoptosis was determined by flow cytometry with annexin V-FITC positive cells and Western blot of PARP expression, respectively. The expressions of FOXO1 were detected by Western blot. The transcriptional activity of FOXO1 was determined by luciferase reporter assay. siRNAs specifically inhibiting FOXO1 were used, and flow cytometry and Western blot were executed to test the role of FOXO1 in CPT-induced apoptosis. Results: CPT was extremely effective in inducing apoptosis of Y79 retinoblastoma cells. FOXO1 was highly expressed in Y79 cells. CPT not only elevated the FOXO1 dephosphorylation level but also promoted its transcriptional activity, suggesting that the activation of FOXO1 was, at least in part, triggered by CPT. The decreased annexin V positive cells and less PARP cleavage demonstrated that siRNAs-mediated inhibition of FOXO1 significantly abrogated CPT-induced apoptosis, indicating that FOXO1 plays an important role in CPT-induced apoptosis. Moreover, the expression of Bim was also elevated with the treatment of CPT, which is in accordance with the activation of FOXO1. Conclusions: Our study provides the evidence that a high level of endogenous FOXO1 expression in retinoblastoma cells contributes, at least in part, to CPT-induced apoptosis, which may help broad application of CPT in retinoblastoma therapy in the future.