Dysregulation of microRNAs (miRs) can contribute to cancer development and progression. In the present study, the function and underlying molecular mechanisms of miR-320 in breast cancer tumorigenesis and progression were investigated. The results of a reverse transcription-quantitative polymerase chain reaction analysis demonstrated that miR-320 was frequently downregulated in breast cancer tissues compared with adjacent normal tissues. In addition, knockdown of miR-320 in breast cancer cell lines promoted cell proliferation and invasion in vitro, whereas miR-320 overexpression had the opposite effect. Furthermore, a Dual-Luciferase reporter assay indicated that SRY-box 4 (SOX4) is a direct target of miR-320, and the restoration of SOX4 in miR-320-overexpressing cells attenuated the tumor-suppressive effects of miR-320. Collectively, these results indicated that miR-320 acts as a tumor suppressor in breast cancer tumorigenesis and progression.