Fisetin, a natural flavonoid found in a variety of edible and medical plants, has been suggested to inhibit the proliferation of various tumor cells and to induce apoptosis. However, the effects of fisetin on breast cancer have rarely been reported and the underlying mechanism is still undefined. The present study explored the anti-cancer effects of fisetin on mammary carcinoma cells and the underlying mechanisms. Following treatment with fisetin, viability of 4T1, MCF-7 and MDA-MB-231 cells were measured by MTT assay. The inhibitory effects of fisetin on proliferation, migration and invasion were evaluated in 4T1 cells using proliferation array, wound-healing assay, and HUV-EC-C-cell barrier based on electrical cell-substrate impedance sensing platform. Cell apoptosis was analyzed by flow cytometry, and western blotting analysis was performed to identify target molecules. A 4T1 orthotopic mammary tumor model was used to assess the fisetin-inhibition on tumor growth in vivo. Test kits were used to examine the liver and kidney function of tumor-bearing mice. The results suggest that fisetin suppressed the proliferation of breast cancer cells, suppressed the metastasis and invasiveness of 4T1 cells, and induced the apoptosis of 4T1 cells in vitro. The potent anti-cancer effect of fisetin was associated with the regulation of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway. In vivo experiments demonstrated that fisetin suppressed the growth of 4T1 cell-derived orthotopic breast tumors and enhanced tumor cell apoptosis, and the evaluated alanine amino transferase and aspartate amino transferase levels in serum of tumor-bearing mice suggested that fisetin may lead to side effects on liver biochemical function. The present study confirms that fisetin exerted an anti-mammary carcinoma effect. However, in vivo experiments also revealed that fisetin had low solubility and low bioavailability. Further investigation is required to determine the clinical value of fisetin.