Abstract: Objective To observe the killing effect of Herceptin and adriamycin sequentially applied on breast cancer cell line in vitro. Methods BT-474 human breast cancer cells in exponential growth phase were treated with Herceptin alone, adriamycin alone and their sequential administration (Herceptin before adriamycin and vice versa), respectively. Under optical microscope, the morphological changes of the cells were observed before and after drug administration. The expression rate and mean fluorescence intensity (MFI) of HER-2/neu and cell death rate were detected by flow cytometry. Results Microscopically, the cells treated with different protocols all exhibited such changes as darkening and increase of cellular debris with irregular cell morphology. Flow cytometry revealed no significant difference in the expression rate of HER-2/neu in each group before and after treatment, but the MFI of HER-2/neu and death rate of the treated cells were significant different from those of the control group (P<0.05). The cell death rate of Herceptin-pretreated cells was significantly higher than that of adri-amycin-pretreated ones (P<0.05). Conclusion Herceptin pretreatment enhances the killing effect of adriamycin on breast cancer cell line BT-474, which provides experimental evidence for designing clinical sequential biochemothrapy of breast cancer.