Osteosarcoma is a malignant bone tumor in which the survival rate is still low. MicroRNA-326 (miR-326) has been proved a potential diagnostic and prognostic marker for several tumors. However, the clinical value of miR-326 is still unknown. In the present study, we detected the expression of miR-326 in the serum of osteosarcoma patients and in osteosarcoma tissues using qRT-PCR. We compared the serum expression of miR-326 with the clinicopathological characteristics and survival of osteosarcoma patients. Finally, we explored the role of miR-326 of the invasion of osteosarcoma tumor cells using cell migration and invasion assays. We found that the expression of miR-326 was significantly decreased in the serum of osteosarcoma patients and osteosarcoma tumor cells compared to healthy controls (P<0.01). Moreover, a receiver operating characteristic (ROC) curve analysis is indicated that serum miR-326 is a potential diagnostic marker of osteosarcoma with an area under the ROC curve of 0.817. Importantly, patients with a lower expression of miR-326 tended to have distant metastasis (P<0.05) and a more advanced clinical stage (P<0.05). In addition, the survival time of patients with depressed miR-326 expression was significantly shorter compared to patients with high miR-326 expression (P<0.05). Further-more, we found that miR-326 could inhibit the proliferation, migration and invasion of osteosarcoma cells. Thus, we demonstrated that miR-326 might be related to the metastasis of osteosarcoma and could be used as a potential diagnostic and prognostic biomarker in osteosarcoma.