Osteosarcoma is a common type of bone tumor that primarily occurs in children and young adults. MicroRNA (miRNA/miR) dysregulation is associated with the progression of osteosarcoma; therefore, the aim of the present study was to investigate the biological functions and molecular mechanisms of miR‑145‑5p in osteosarcoma. The expression of miR‑145‑5p in osteosarcoma tissues and cell lines was quantified using reverse transcription‑quantitative PCR (RT‑qPCR). The effect of miR‑145‑5p on the proliferation of osteosarcoma cells was detected using Cell Counting Kit‑8 and colony formation assays, as well as cell cycle distribution analysis. The effect of miR‑145‑5p on tumor growth was further investigated in vivo using a subcutaneous tumor model in nude mice. The interaction between miR‑145‑5p and E2F transcription factor 3 (E2F3) was determined using bioinformatics analysis, a luciferase assay, RT‑qPCR and western blotting. The results revealed that miR‑145‑5p expression was decreased in osteosarcoma cell lines and tissues compared with the corresponding normal controls. Increased miR‑145‑5p expression inhibited the proliferation and colony formation ability of osteosarcoma cells, and induced G1 phase arrest. Furthermore, mice injected with tumor cells overexpressing miR‑145‑5p exhibited smaller tumors than those in the control group. Further investigation revealed that miR‑145‑5p binds to and decreases the expression of E2F3. In addition, the mRNA levels of E2F3 were negatively associated with miR‑145‑5p in osteosarcoma tissues, and increasing E2F3 expression abrogated the inhibitory effects of miR‑145‑5p on osteosarcoma cells. Collectively, the results obtained in the present study suggest that miR‑145‑5p may suppress the progression of osteosarcoma, and may serve as a useful biomarker for the diagnosis of osteosarcoma, as well as a therapeutic target.