Background: Breast cancer is the most common malignancy in women worldwide, so it is imperative to find new medications for this disease. The dry seeds of Brucea javanica have been extensively used in Traditional Chinese Medicine (TCM) for the treatment of tumor. Objectives: To isolate antitumor peptides from B. javanica seeds and research their antitumor activity with MCF-7 cells. Materials and Methods: Globulin of B. javanica was extracted and enzymatically hydrolyzed by pepsin. Then ultrafiltration, gel filtration chromatography (GFC), and reverse-phase high-performance liquid chromatography (RP-HPLC) were employed to purify the peptides. Anticancer activity was studied in humans in human breast cancer MCF-7 cells, MTT (3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)-assessed cell viability, Giemmsa staining determined whether the cells should undergo apoptosis, and apoptosis and cycle assays assessed the antitumor mechanism of the cells. Real time quantitative polymerase chain reaction (RT-qPCR) was used to study the alterations of oncogenes and cancer metastasis suppressor genes in MCF-7 cells treated with peptide extracts. Results: The fraction F9-9 extracted from B. javanica significantly inhibited the proliferation of MCF-7 cells with the IC50 of 0.124 ± 0.004 μg/mL. The possible mechanisms of this fraction were further explored. F9-9 obviously induced cell cycle arrest at G0/G1 phase and promoted apoptosis of MCF-7 cells. The underlying molecular mechanisms were explored by RT-qPCR and results showed that in B. javanica small molecular peptides upregulated the expression of tumor suppressor genes P53 and PTEN as well as tumor metastasis suppressor gene NM23H-1. Conclusion: This study suggests that B. javanica peptides have therapeutic activity and mechanisms for antibreast cancer, encouraging further isolation and purification to obtain monomeric peptides of this extract for anticancer use.