Retinoblastoma (RB) is the most frequent primary intraocular cancer. It has been demonstrated by previous studies that retinoblastoma is initiated primarily by the inactivation of the retinoblastoma Rb1 gene in retinal cells. However, additional genetic alterations than Rb1 mutation could play important roles in the process of transforming benign retinal cells into retinoblastoma tumor cells. In this study, we identified that microRNA miR-433 is one of such genetic factors. We found that the expression levels of miR-433 were downregulated in RB tissues. We also determined that miR-433 negatively regulated RB cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis of RB cells. We used bioinformatics method to predict and confirmed that Notch1 and PAX6 were miR-433 target genes in RB cells. Importantly, we demonstrated that restoration of Notch1 and PAX6 expression partially rescued the inhibition of cell proliferation and metastasis induced by miR-433 overexpression, suggesting that miR-433 regulates RB cell proliferation and metastasis through suppressing the expression of Notch1 and PAX6.