The present study aimed to explore the expression of Annexin A3 (ANXA3) in breast cancer cells and the mechanisms involved in the regulatory effects of ANXA3 on proliferation, invasion and migration of breast cancer cells. Fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were used to measure the expression of ANXA3 mRNA and protein in two breast cancer cell lines (MDA-MB-231 and MCF-7). Three ANXA3 silencing shRNA plasmids (ANXA3-sh1-3) and one negative control plasmid were constructed, and the Lipofectamine transfection method was used for transfecting human breast cancer cell line MDA-MB-231. Flow cytometry was used to measure the transfection efficiency. The expression of ANXA3 protein was measured by western blotting. Cell cycle distribution and apoptosis were assessed by flow cytometry. Migration and invasion of the transfected cells were evaluated using wound healing and Transwell assays, respectively. The expression levels of ANXA3 mRNA and protein were significantly higher in the MDA-MB-231 cells than levels in the MCF-7 cells. Western blotting showed that the ANXA3 protein level was significantly lower in the MDA-MB-231-Sh cells than that in the MDA-MB-231 and MDA-MB-231-NC cells. In addition, the percentage of G(0/1) cells and the apoptosis rate were significantly higher, while the cell proliferation rate was significantly lower, in the MDA-MB-231-Sh cells when compared with the MDA-MB-231-NC and MDA-MB-231 cells. The cell migration and invasion abilities were also lower in the MDA-MB-231-Sh cells than these abilities in the MDA-MB-231-NC and MDA-MB-231 cells. The present study investigated the relationships between ANXA3 and proliferation, apoptosis, migration and invasion of breast cancer cells to elucidate the mechanisms involved in the development, progression, invasion and metastasis of breast cancer.