Accumulating evidence indicates that microRNAs are involved in multiple processes in cancer development and progression, and several miRNAs have emerged as candidate components of oncogene or tumor-suppressor networks in retinoblastoma. miR-101 has been identified as a tumor suppressor in several types of human cancer. However, the specific function of miR-101 in retinoblastoma remains unclear. In the present study, we found that the expression of miR-101 in retinoblastoma tissues was much lower than that in the normal controls. In addition, downregulation of miR-101 more frequently occurred in retinoblastoma specimens with adverse clinicopathological and histopathological features. In addition, miR-101 inhibited cell viability and progression in retinoblastoma cells by promoting cell apoptosis and arresting the cell cycle. Finally, we found that miR-101 directly inhibited EZH2 expression by targeting its 3'-UTR, and EZH2 was upregulated and inversely correlated with miR-101 expression in the retinoblastoma tissues. Thus, for the first time, we provide convincing evidence that downregulation of miR-101 is associated with tumor aggressiveness in retinoblastoma and inhibits cell growth and proliferation of retinoblastoma cells by targeting EZH2. In conclusion, all the evidence supports the tumor-suppressor role of miR-101 in human retinoblastoma.