Moesin-ezrin-radixin-like protein (Merlin) has been identified as a tumor suppressor in several types of cancers. However, the biological function of Merlin in osteosarcoma remains unclear. MicroRNAs (miRNAs) can influence cancer progression by targeting oncogenes or anti-oncogenes. In this study, we sought to evaluate the regulation of Merlin expression by miR-25-3p and the role of the miR-25-3p/Merlin axis in osteosarcoma progression, with the aim of identifying a potential therapeutic target for osteosarcoma. Materials and Methods: TCGA (The Cancer Genome Atlas) database was used to analyze the correlation between Merlin expression and prognosis. RT-qPCR and Western blotting analyses were performed to compare Merlin expression between normal and malignant cells. A dual-luciferase reporter assay was performed to evaluate the direct targeting of Merlin by miR-25-3p. We overexpressed miR-25-3p, or/and Merlin, in U-2 OS and 143B cells, and studied their cellular functions in vitro. MTT and colony formation assays were performed to determine the effects on cell growth. EdU and cell cycle assays were performed to analyze the effects in cell replication. We used annexin V-fluorescein isothiocyanate and propidium iodide to stain apoptotic cells, and analyzed the cells using flow cytometry. The effects on cell metastasis were studied in wound healing and transwell assays. Lastly, the underlying mechanism was determined in RT-qPCR and Western blotting experiments. Results: Low Merlin expression was linked to poor prognosis. miR-25-3p was observed to directly target Merlin and downregulate its expression. miR-25-3p promoted cell growth, migration, and invasion, and inhibited apoptosis induced by cisplatin. Moreover, the overexpression of Merlin reversed the abovementioned effects of miR-25-3p. Further, the miR-25-3p/Merlin axis was observed to play an important role in the Hippo pathway, and regulated the expression of genes such as BIRC5, CTGF, and CYR61. Conclusion: miR-25-3p functions as an oncogenic microRNA in osteosarcoma by targeting Merlin, and may serve as a potential therapeutic target for osteosarcoma.