Osteosarcoma harbors highly metastatic properties, accounting for postoperative recurrence and metastasis. Actin-like protein 6A (ACTL6A) regulates cell proliferation, migration and differentiation. However, the biologic role of ACTL6A in osteosarcoma remains unknown. In this study, the results showed that, by analysis of frozen fresh primary tumor tissues, matched non-cancerous bone tissues (NCBTs) and biopsy lung metastatic nodule tissues from 30 osteosarcoma patients after radical surgical resection, ACTL6A was overexpressed in osteosarcoma tissues compared with matched NCBTs, and its expression level was associated with osteosarcoma metastasis. Immunohistochemical analyses of osteosarcoma tissue samples from two independent cohorts of formaldehyde-fixed, paraffin-embedded osteosarcoma tissue samples from total of 186 osteosarcoma patients showed that high ACTL6A expression correlated with malignant clinicopathological features such as larger tumor size, high Ennecking grade, high histologic grade, and advanced tumor node metastasis stage. High ACTL6A expression was associated with poor prognosis for patients with osteosarcoma, and an independent and significant risk factor for disease-free survival and overall survival after radical tumor resection. Both in vitro and in vivo assays showed that ACTL6A overexpression promoted osteosarcoma cell invasion and metastasis, whereas knockdown of ACTL6A expression reduced osteosarcoma cell malignant behavior such as invasion and metastasis. Furthermore, we proved that ACTL6A promoted osteosarcoma cells of metastasis through facilitating epithelial-mesenchymal transition (EMT). In conclusion, data from the present study demonstrated that ACTL6A was associated with poor survival and promoted osteosarcoma cell metastasis through EMT, suggesting that ACTL6A may be a novel prognostic biomarker and therapeutic target for osteosarcoma.