Long non-coding RNA (lncRNAs) DLX6-AS1 plays significant roles in various types of malignant tumors, including osteosarcoma (OS), the most prevalent primary malignant bone tumor. However, the role and mechanism of DLX6-AS1 have not been fully illuminated in OS. Here, we aimed to find a novel mechanism for DLX6-AS1 in regulating the development of OS through sponging microRNA (miRNA). According to the luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assay, miRNA (miR)-141-3p can physically interact with DLX6-AS1 and Rab10. The expressions of DLX6-AS1 and Rab10 were upregulated and miR-141-3p was downregulated in OS tissues and cells (MG-63 and U2OS), as described by RT-qPCR and western blotting. Moreover, there was a negative correlation between the expression of miR-141-3p and either DLX6-AS1 or Rab10, and a positive correlation between DLX6-AS1 and Rab10. Functionally, cell proliferation, migration and invasion were evaluated by utilizing the MTT assay and transwell assays. As a result, DLX6-AS1 knockdown suppressed OS cell proliferation, migration and invasion in MG-63 and U2OS cells, which was abolished by the downregulation of miR-141-3p. Similarly, the upregulation of Rab10 not only promoted OS cell progression in vitro, but also blocked the inhibitory effect of miR-141-3p overexpression in OS cells. Notably, DLX6-AS1 knockdown could, in turn, reverse the promoting effect of Rab10 on OS cell progression. Xenograft experiments depicted that DLX6-AS1 knockdown restrained the tumor growth of MG-63 cells in vivo. In conclusion, the knockdown of DLX6-AS1 might suppress OS progression via sponging miR-141-3p and downregulating Rab10, suggesting a novel DLX6-AS1/miR-141-3p/Rab10 pathway in OS progression.