microRNAs are small, highly conserved, non‐coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition, and are widely involved in carcinogenesis and cancer development. In this study, the expression profile of microRNA‐133a (miR‐133a) was examined in breast cancer cells and breast cancer tissues. The results showed that expression of miR‐133a in both breast cancer cells and breast cancer tissues was significantly down‐regulated. Over‐expression of miR‐133a in tumor cells arrested the cell cycle by drastically decreasing the G₂/S phase and retarded the newly synthesized DNA, suggesting a regulatory role for miR‐133a in proliferation of breast cancer cells. Bioinformatics prediction showed that epidermal growth factor receptor (EGFR) is a potential target for miR‐133a. A dual luciferase reporter gene assay showed that miR‐133a bound to the 3′ UTR of EGFR but not a mutated 3′ UTR, thereby down‐regulating the protein expression level. Accordingly, we found that expression of EGFR protein decreased with increased expression of miR‐133a in MCF‐7 and MDA‐MB‐231 cells. Over‐expression of miR‐133a in breast cancer cells resulted in suppression of the level of phosphorylated Akt protein (p‐Akt) and inhibition of p‐Akt nuclear translocation. These results demonstrate that miR‐133a, which may act as a tumor suppressor in breast cancer, regulates the cell cycle and proliferation in tumorigenesis by targeting EGFR through the downstream signal molecule Akt. Overall, these results show that miR‐133a may be used as biomarker and/or therapeutic target for diagnosis and therapy of breast cancer.