Osteosarcoma (OS) is among the most common primary tumors of bone tissue, and occurs primarily in children and young adults. Despite the development of novel therapeutic approaches, the prognosis of OS remains poor. MicroRNAs (miRNAs) are involved in the development and progression of various types of human cancer and may have potential as novel therapeutic targets for cancer treatment. The present study aimed to investigate the expression and biological functions of miRNA‑25‑3p in OS, and explore the molecular mechanisms underlying its actions. In the present study, miRNA‑25‑3p was detected in OS tissues and cell lines. The functional roles of miRNA‑25‑3p in OS cells were evaluated using a Cell Counting Kit 8 assay and cellular migration and invasion assays. The molecular mechanisms underlying the tumor‑suppressing roles of miRNA‑25‑3p in OS were explored using bioinformatics analysis, luciferase reporter assay, western blotting and the reverse transcription‑quantitative polymerase chain reaction. The expression of miRNA‑25‑3p was revealed to be downregulated in OS tissues and cell lines compared with non‑tumor bone tissues and normal osteoblasts, respectively. miRNA‑25‑3p overexpression was demonstrated to significantly suppress the proliferation, migration and invasion of OS cells in vitro. In addition, sex‑determining region‑related high mobility group box (SOX) 4 was identified as a direct target gene of miRNA‑25‑3p, and was further investigated. Similarly to miRNA‑25‑3p overexpression, SOX4 knockdown was demonstrated to suppress OS cell proliferation, migration and invasion. Furthermore, SOX4 expression was revealed to be significantly upregulated in OS tissues compared with in adjacent non‑tumor bone tissues, and Spearman's correlation analysis indicated a negative correlation between SOX4 mRNA and miRNA‑25‑3p expression levels in OS tissues. The present findings suggested that miRNA‑25‑3p may act as a tumor suppressor by targeting SOX4 expression in bone tissue. Therefore, miRNA‑25‑3p may have potential as a novel therapeutic target for the treatment of patients with OS.