ERBB2 overexpression in estrogen receptor (ER)-positive breast cancer cells such as BT474 (BT) cells has been found to confer resistance to tamoxifen, and suppression of ERBB2 improves the antiproliferative effects of tamoxifen. In this study, the responsiveness to tamoxifen in the BT/HerR, Herceptin-resistant BT cell lines established through constant Herceptin exposure, was evaluated. Compared with BT cells, improvement of sensitivity to tamoxifen in BT/HerR was demonstrated by ER functional analysis and cell proliferation assay. Tamoxifen in the resistant cell line was found to inhibit 17beta-estradiol-stimulating estrogen-responsive gene pS2 expression more effectively than in BT cells in real-time PCR assay. Western blot analysis showed that cross-phosphorylation between ER and downstream components of ERBB2 was attenuated in BT/HerR cells. ER redistribution from cytoplasm to nucleus could be found in these cells through immunofluorescence and confocal studies, and importantly, chromatin immunoprecipitation studies demonstrated that tamoxifen induced occupancy of the pS2 promoter by ER and nuclear receptor corepressor (NCOR1) instead of coactivator NCOA3 in these cells. Finally, combination of tamoxifen and Herceptin was found to improve the sensitivity of BT/HerR cells to Herceptin. Our results suggest that the ER genomic pathway in the ER-positive and Herceptin-resistant breast cancer cells may be reactivated, allowing tamoxifen therapy to be effective again, and a combination of tamoxifen and Herceptin can be a potential therapeutic strategy for ER-positive and Herceptin-resistant human breast cancer.