Objective To explore the B cell lymphatic factor 9 (BCL9) gene expression in different human breast cancer cell lines. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the mRNA and protein expression levels of BCL9 in human breast cancer cell lines MDA-MB-231, MCF-7 and MDA-MB-453. Flow cytometry was used to detect the cell cycle, proliferation index was calculated, and proliferative ability was compared among different cell lines. Results Real-time PCR revealed that the relative expression of BCL9 was highest in MDA-MB-231 cells (0.016±0.004) followed by MCF7 cells (0.008±0.002), lowest in MDA-MB-453 cells (0.004±0.002) (Comparison between MDA-MB-231 and MCF-7, P=0.000; Comparison between MDA-MB-231 and MDA-MB-453, P=0.000; Comparison between MCF-7 and MDA-MB-453, P=0.021). The relative expression of BCL9 protein was highest in MDA-MB-231 cells (0.629±0.101), followed by MCF-7 cells (0.204±0.990), and lowest in MDA-MB-453 cells (0.397±0.196) with the difference being statistically significant (Comparison between MDA-MB-231 and MCF-7, P=0.012; Comparison between MDA-MB-231 and MDA-MB-453, P=0.000; Comparison between MCF-7 and MDA-MB-453, P=0.030). Flow cytometry indicated that the proliferation index in MDA-MB-231 cells (0.483±0.010) was significantly higher than in MDA-MB-453 cells (0.358±0.032, P=0.009) and MCF-7 cellls (0.397±0.061, P=0.040). The expression of BCL9 gene was positively correlated with the expression of BCL9 (P=0.000); BCL9 gene was positively correlated with cell proliferation index (P=0.040). Conclusion The expression level of BCL9 in human breast cancer cell lines was different, however, for the three negative breast cancer, the MDA-MB-231 cell line showed a higher proliferation ability. The expression level of BCL9 was also higher, and both were positively correlated. Key words: Breast cancer; B cell lymphatic factor 9; MDA-MB-231; MDA-MB-453; MCF-7