Objective: Breast cancer remains the most common cancer in women, worldwide. It has been shown that microRNAs play essential roles in tumorigenesis and progression in various cancers, including breast cancer. The current study assessed the roles of miR-106a-5p in proliferation, chemotherapy sensitivity, migration, and invasion of breast cancer cells. Methods: MTT assays were performed to examine the proliferation of MCF-7 and MDA-MB-231 breast cancer cells with altered expression of miR-106a-5p, with or without 5-fluorouracil treatment. Transwell migration and Matrigel invasion assays were used to assess migration and invasion of MCF-7 and MDAMB-231 breast cancer cells. Western blotting was performed to examine protein expression levels. Caspase-Glo3/7 assays were used to examine the effects of miR-106a-5p on 5-fluorouracil-induced apoptosis in MCF-7 and MDAMB-231 breast cancer cells. Results: Forced expression of miR-106a-5p improved proliferation, migration, and invasion of breast cancer cells. Moreover, miR-106a-5p decreased the sensitivity of MCF-7 and MDA-MB-231 breast cancer cells after 5-fluorouracil treatment. Additionally, 5-flurouracil-induced apoptosis in breast cancer cells was reduced by miR-106a-5p by regulation of BAX and Bcl-2 expression. Furthermore, miR-106a-5p regulated epithelial to mesenchymal transition (EMT) by altering expression of E-cadherin, N-cadherin, Snail, and VIMENTIN IN BOTH MCF-7 and MDA-MB-231 breast cancer cells. Further examination showed that expression levels of PTEN and p-AKT in MCF-7 and MDA-MB-231 breast cancer cells were changed after aberrant expression of miR-106a-5p. Conclusion: miR-106a-5p plays important roles in tumorigenesis and progression in breast cancer cells. It might act as a potential biomarker, predicting chemotherapy response and prognosis in breast cancer.