Increasing evidence has demonstrated that microRNAs play a critical role in breast cancer (BC) progression. microRNA-101 (miR-101) has been considered a tumor suppressive miRNA in different cancer types. This study aimed to investigate miR-101 expression in BC tissues and to investigate its roles in BC progression that are mediated by Sex-determining region Y-box 2 (SOX2), a critical oncogene in various cancers. qRT-PCR and immunohistochemistry were performed to detect miR-101 and SOX2 expression in BC tissues and paired normal tissues or BC cell lines. MTT, transwell migration, wound healing, colony formation, flow cytometric and xenograft assays were performed to determine the influence of miR-101 and SOX2 on the malignant behaviors of BC cells in vitro and in vivo. miR-101 was significantly downregulated in BC tissues and cell lines. miR-101 overexpression inhibited the malignant behaviors of BC cells, both in vitro and in vivo. miR-101 downregulation had the converse effect. A miR-101 binding site was identified by luciferase reporter assay in the 3’UTR of SOX2. SOX2 was upregulated in BC tissues and cell lines, and its upregulation was associated with lymph node metastasis, pathological grade and TNM classification. SOX2 knockdown mimicked the effects of miR-101 overexpression on the malignant behaviors of BC cells, while SOX2 overexpression mitigated the miR-101-induced inhibition of these effects. Our study revealed that miR-101 plays a critical role in suppressing tumor progression by directly targeting SOX2.