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Introduction Annexin A3 (ANXA3) participates in various tumor-associated biological processes, including tumor initiation, progression, and metastasis. The present study was designed to investigate the expression and function of ANXA3 in breast cancer cells. Materials and Methods Annexin A3 protein expression in breast cancer cell lines was evaluated using Western blot analysis. ANXA3 expression in MDA-MB 231 breast cancer cells was silenced by RNA interference, and the effects of RNA silencing on cell proliferation, colony forming ability, wound-healing, and invasiveness were evaluated. Levels of ANXA3 expression in 30 primary breast cancers were assayed using immunohistochemistry and correlated with patient survival. Results Levels of ANXA3 expression were higher in the basal subtype of breast cancer cells, such as MDA-MB 231, HCC-70, and HCC-1954 cells, than in other subtypes. ANXA3 silencing inhibited the activities of MDA-MB 231 and HCC-1954 cells, including their proliferation, invasion across transwell membranes, and wound-healing and colony forming abilities. ANXA3 small interfering RNA (siRNA) also reduced the expression of cycle-dependent kinase protein and increased the expression of E2F1 and p27 proteins compared with control siRNA. Expression of ANXA3 was closely correlated with tumor size, with higher ANXA3 expression associated with reduced disease-free survival in breast cancer patients. Conclusion These findings indicate that ANXA3 is associated with the natural progression of breast cancer and might be a potential prognostic marker of patient survival.