MicroRNA (miR)-320a is specific to vertebrates and has been indicated to serve a role in a number of cancer types, such as gastric, colorectal, pancreatic and ovarian cancer. miR-320a has been reported to be expressed at high levels in retinoblastoma tissues; however its role and mechanism of function in retinoblastoma remain to be elucidated. The aim of the present study was to investigate the role of miR-320a in retinoblastoma cells and the underlying mechanisms. The expression of miR-320a in retinoblastoma cell lines Y79 and WERI-Rb-1, and normal human retinal pigment epithelial cell line ARPE-19 was examined via reverse transcription-quantitative PCR (RT-qPCR). TargetScan bioinformatics analysis and dual-luciferase reporter assay were used to predict and reveal the target gene of miR-320a. Target gene expression was detected via RT-qPCR in retinoblastoma cell lines and ARPE-19 cells. Subsequently, gain-or loss-of-function experiments for miR-320a and tumor suppressor candidate 3 (TUSC3) were performed to study the role of miR-320a/TUSC3 in retinoblastoma cells. Cell viability and apoptosis were assessed via MTT and flow cytometry analysis, respectively. Compared with ARPE-19 cells, miR-320a was prominently expressed in retinoblastoma cell lines. TUSC3 was predicted to be a target gene of miR-320a. Compared with ARPE-19 cells, the expression of TUSC3 in retinoblastoma cell lines was reduced. The results of MTT and flow cytometry analysis revealed that overexpression of TUSC3 reduced the viability of retinoblastoma cells and induced apoptosis. Additional analysis indicated that miR-320a inhibitor enhanced the expression of the target gene TUSC3, thereby inhibiting retinoblastoma cell viability and inducing apoptosis. The effects of miR-320a inhibitor on retinoblastoma cells were inhibited by TUSC3-short hairpin RNA. miR-320a regulated the viability and apoptosis of retinoblastoma cells via targeting TUSC3. Therefore, the present study provided a reference for investigating a potential target for the clinical treatment of retinoblastoma.