Abstract. MicroRNAs (miRs) have been demonstrated to be associated with multiple processes in the development and progression of human malignancies. Previous studies have observed aberrant downregulation of miR-144 in several types of cancer, including osteosarcoma. However, the function of miR-144 and the underlying mechanism in osteosarcoma remain to be elucidated. The present study indicated that miR-144 was markedly downregulated in osteosarcoma tissues and cell lines compared with that in the normal controls. Restoration of miR-144 significantly inhibited cell proliferation, migration and invasion of MG-63 osteosarcoma cells. In addition, Rho-associated coiled-coil containing protein kinase 1 (ROCK1) was identified as a novel target of miR-144 in MG-63 osteosarcoma cells. Furthermore, knockdown of ROCK1 suppressed the proliferation, migration and invasion of MG-63 osteosarcoma cells to a similar extent to the effects of miR-144 overexpression. In addition, the mRNA expression of ROCK1 was increased in osteosarcoma tissues and was negatively correlated with the expression of miR-144. In conclusion, the results of the present study suggested that miR-144 acts as a tumor suppressor by targeting ROCK1 in osteosarcoma.