Osteosarcoma (OS), derived from the primitive bone-forming mesenchymal cells in the long bones, is the leading cause of cancer-related deaths in adolescents and children. MiRcroRNA-139 (miR-139) has been reported to be abnormally expressed and play important roles in several cancer types. However, the expression pattern, detailed biological roles and associated molecular mechanisms of miR-139 in OS have remained to be fully elucidated. This study showed that miR-139 expression was downregulated in OS tissues and cell lines. Upregulation of miR-139 significantly inhibited cell proliferation and invasion in OS. Bioinformatics analysis predicted that ROCK1 is a potential target of miR-139. Luciferase reporter assay demonstrated that miR-139 can directly interact with the 3’-untranslated region of ROCK1. RT-qPCR and Western blotting analysis demonstrated that miR-139 had regulatory effects on endogenous ROCK1 expression in OS. Furthermore, ROCK1 knockdown significantly produced effects similar to those induced by miR-139 overexpression in OS cells. Therefore, the miR-139/ROCK1 axis may be a potential target for novel potential therapeutic strategies for treatment of OS.