MicroRNAs are increasingly recognized as important regulators of cancer. The aim of the present study was to investigate the role of miR-106b in the regulation of Y79 retinoblastoma. Y79 cells were transfected with antisense oligonucleotides (ASO) against miR-106b (ASO-miR-106b) or ASO-control. After transfection, the levels of miR-106b were monitored with real-time PCR (RT-PCR). The effects of ASO-miR-106b transfection on cell viability was evaluated by Cell Counting Kit-8 (CCK-8) analysis at 24, 48 and 72 h after transfection. Subsequently, the cells were stained with Annexin V-FITC and propidium iodide (PI) and subjected to flow cytometry to assess cell apoptosis. Transwell assay was used to analyze cell migration. Changes in Runt-related transcription factor 3 (Runx3) mRNA and proteins levels were also evaluated. miR-106b was downregulated by ASO-miR-106b at 48 and 72 h after transfection, accompanied by a decrease in cell viability and proliferation, as well as an increase in cell apoptosis. Transwell analysis indicated that cells treated with ASO-miR-106b exhibited significantly lower cell migratory abilities. The mRNA and protein level of Runx3 were upregulated after transfection. These results demonstrated that suppression of miR-106b inhibited Y79 cell proliferation and migration. The upregulation of Runx3 after miR-106b suppression ascertained that Runx3 is a tumor-suppressor in retinoblastoma and is a target of miR-106b.