Osteosarcoma (OS) is the most common bone tumor in children and young adults, and is an aggressive tumor with poor prognosis. MicroRNAs (miRNAs) are aberrantly expressed in various types of cancer, and contribute to cancer tumorigenesis and progression. In the present study, the potential prognostic value and biological function of miRNA-136 (miR-136) in OS was investigated. Reverse transcription-quantitative polymerase chain reaction analysis was used to evaluate the expression of miR-136 in OS tissues and cell lines. Kaplan-Meier survival analysis and Cox regression analysis were conducted to investigate the prognostic significance of miR-136. Various in vitro cell based assays were used to evaluate the effects of miR-136 on the biological behavior of OS cells. A luciferase assay was performed to determine the key miR-136 targets associated with OS. The expression of miR-136 was significantly downregulated in osteosarcoma tissues and cells compared with the normal controls (all P<0.05). Decreased miR-136 expression was significantly associated with Enneking staging (P=0.030) and distant metastasis (P=0.016). Decreased miR-136 expression in patients was associated with shorter overall survival compared with patients with increased expression levels (log-rank test; P<0.05). The expression of miR-136 was indicated as an independent prognostic factor for the patients (hazard ratio= 0.496; 95% confidence interval=0.250-0.987; P=0.046). MTT, transwell and Matrigel assays demonstrated that upregulation of miR-136 decreased proliferation, migration and invasion of OS cells. Bioinformatics and luciferase assays demonstrated that migration and invasion enhancer 1 (MIEN1) is a direct target of miR-136. Together, the results suggested that miR-136 functions as a tumor suppressor gene to regulate proliferation, migration and invasion of OS cells. MIEN1 was a potential target of miR-136. Additionally, miR-136 may serve as a prognostic biomarker for OS.