Abstract: To investigate the effects and mechanism of action of herceptin on Notch-1 protein in breast cancer SK-BR3 cells, and to explore the significance of Notch-1 signaling pathway in breast cancer cell resistance to herceptin. Methods: The breast cancer cells SK-BR3 with HER2 overexpression and MDA-MB-231 overexpression with HER2 non-overexpression were selected. Immunocytochemistry and Western-blot analysis were used to detect the expression of Notch-1 protein and activate Notch-1 ( Notch-1IC ) in the SK-BR3 and MDA-MB-231 cells, respectively. SK-BR3 cells were treated with herceptin. Western blot analysis was used to detect the expression of Notch-1IC and HER2 proteins. Reverse transcriptase polymerase chain reaction was used to assay HES-1 mRNA expression. Co-immunoprecipitation detected the interaction between HER2 and Notch-1 proteins in the SK-BR3 cells. Results: The level of Notch-1IC nucleic localization and the expression of Notch-1IC protein in SK-BR3 cells ( 9.37 ± 0.64 ) were significantly lower than in the MDA-MB-231 cells ( 21.665 ± 1.11 ) ( P < 0.01 ). SK-BR3 cells were treated with herceptin. Compared with the controls, the expression of Notch-1IC protein and HES-1 mRNA was significantly increased ( P < 0.01 ). There was no apparent change in HER2 protein expression after herceptin treatment ( F = 0.973, P > 0.05 ). Co-immunoprecipitation showed coprecipitation between the Notch-1 and HER2 proteins. Conclusion: The activity of Notch-1 protein was decreased in the breast cancer cells with HER2 overexpression. HER2 could combine with Notch-1, thereby negatively regulating Notch-1 activity. Herceptin could increase the activity of Notch-1, which could be associated with the cell resistance.