MicroRNA-338-3p (miR‑338-3p), a recently discovered miRNA, has been reported to be downregulated and play tumor suppressor roles in gastric cancer, ovarian cancer, colorectal carcinoma and lung cancer by targeting several genes. However, the role and potential mechanism of miR‑338-3p in breast cancer (BC) is still unclear. In the present study, we investigated the roles and mechanisms of miR‑338-3p in human breast cancer. miR‑338-3p expression was determined by qRT-PCR in human BC cell lines, and clinical significantly of miR‑338-3p expression was further evaluated. Furthermore, the function of miR‑338-3p in breast cancer also was investigated by several in vitro approaches and in nude mouse models. Luciferase assay and western blot analysis were performed to validate the potential targets of miR‑338-3p after the preliminary screening by employing open access software. It was found that miR‑338-3p was significantly downregulated in both BC tissues and cell lines and the low expression of miR‑338-3p was inversely correlated with lymph node metastatic and TNM stage status (P<0.01). Function assay showed that the overexpression of miR‑338-3p in BC cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell apoptosis and cell cycle arrest at G1/G0 stage, as well as suppressed tumor growth in the nude mouse model. Luciferase assay and western blot analysis identified sex-determining region Y-box 4 (SOX4) as a direct and functional target of miR‑338-3p. These findings revealed that miR‑338-3p may act as a tumor suppressor in breast cancer by targeting SOX4, suggesting miR‑338-3p as a novel strategy for breast cancer treatment.