Objective: To analyze the regulatory mechanism of miR-136 on the biological behavior of osteosarcoma cells, and reveal the role and mechanism of miR-136 in osteosarcoma. Methods: The expressions of miR-136 and ITGAV in OS tissues and adjacent normal tissues were detected. TargetScan, Dual-Luciferase Reporter Assay, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to confirm whether ITGAV was the target gene of miR-136. The effects of miR-136 and ITGAV on the proliferation, cycle, and migration of MG63 cells were determined by using CCK-8, flow cytometry and Scratch-wound assay. The mouse model of osteosarcoma was established, and the effect of miR-136 on ITGAV expression was detected by using RT-PCR and Western blotting. Results: We found that the expression of miR-136 in OS tissues was significantly decreased, while the expression of ITGAV was increased. MiR-136 could specifically bind to the 3'-UTR of ITGAV and negatively regulate ITGAV. Adopting cell experiments, cell proliferation, S-phase cell ratio, and migration ability of MG63 and U2OS osteosarcoma cell were increased in ITGAV group, while such ability was inhibited in miR-136 group, and the miR-136+ITGAV group could reverse the inhibitory effects of miR-136 group on MG63 and U2OS cell proliferation, S-phase cell ratio, and migration ability. In nude mouse model, miR-136 could inhibit osteosarcoma development, and ITGAV expression was decreased in the miR-136 mimic group as comparing with the control group, but an opposite result occurred in the miR-136 inhibitor group. Conclusion: The abnormal expression of miR-136 may be associated with osteosarcoma. MiR-136 inhibits the biological behavior of osteosarcoma cells and in vivo tumor growth by targeting the expression of ITGAV, suggesting that miR-136 may pose tumor-suppressive effects.