Research into the expression and function of microRNAs (miRNAs/miR) in human cancer has provided novel insights into the molecular mechanisms underlying carcinogenesis and cancer progression. Aberrant miRNA expression has been reported in retinoblastoma (RB) and several other types of human cancer. The present study demonstrated that miR‑320 is significantly downregulated in RB tissues and cell lines. Furthermore, overexpression of miR‑320 was demonstrated to inhibit proliferation, migration and invasion of RB cells. Bioinformatic analysis identified specificity protein 1 (SP1) as a potential target gene of miR‑320. Luciferase reporter assay confirmed that the SP1 3'‑untranslated region contains a direct binding site for miR‑320, and restoration of miR‑320 expression decreased the mRNA and protein expression levels of SP1. Notably, SP1 silencing induced a similar effect on the proliferation, migration and invasion of RB cells as that observed with miR‑320 overexpression, further supporting the hypothesis that SP1 is a direct functional target of miR‑320 in RB. In conclusion, these findings indicate that miR‑320 may be an effective therapeutic target for the treatment of RB.