Osteosarcoma (OS) is a common primary malignancy in adolescents and children. MicroRNAs (miRNAs or miRs) can regulate the progression of OS. Herein, we explored the target genes and effects of miR-9 in OS. Cell growth, colony formation and cell cycle were respectively examined using a cell counting kit-8 (CCK-8), crystal violet staining and flow cytometry. The target gene of miR-9 was predicted according to the MicroRNA.org website. Luciferase activity was examined using a dual luciferase reporter gene assay kit. The corresponding factors levels were analyzed by carrying out reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. A mouse model of OS was also established and the volume and weight of the tumors of the mice with OS were measured. The levels of p16 in the mice with OS were detected by immunohistochemistry (IHC). The data revealed a high expression of miR-9 and a low expression of p16 in the OS tissue. p16 was found to be the target gene for miR-9 in OS. miR-9 depletion decreased the proliferation and colony formation of Saos-2 cells by arresting the cells at the G1 phase, accompanied by the downregulation of cyclin A, cyclin D1 and c-Myc expression levels. Moreover, miR-9 depletion inhibited the phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In vivo , miR-9 depletion decreased the tumor volume and weight and increased p16 expression in the mouse tumor tissues. Nevertheless, p16 silencing reversed the suppressive effects of miR-9 inhibitors on OS cells. On the whole, the findings of this study substantiate that miR-9 depletion suppresses cell proliferation by targeting p16 in OS and by mediating the activation of the ERK/p38/JNK pathway.