The development of resistance to chemotherapeutic and targeted agents occurs commonly in breast and other cancer types, leading to resumption of tumour growth and progression to metastasis, despite ongoing treatment. Processes underlying the loss of sensitivity to previously efficacious drugs are diverse, however elevated expression of cellular efflux transporters that reduce intracellular drug accumulation is frequently associated, either as a primary or supporting mechanism and particularly in cancer stem cells. Androgens, including 5α-dihydrotestosterone (DHT) and Hedgehog signalling inhibitors such as the Smoothened inhibitor, cyclopamine, decrease the proliferation of MCF-7, T-47D and other breast cancer cell lines. PCR array analysis of DHT- and cyclopamine-induced changes in gene expression indicated markedly decreased expression of ABCG2, a cell membrane transporter that exports metabolites including oestrogens, but also transports the chemotherapeutic agents mitoxantrone and doxorubicin and targeted agents including tyrosine kinase inhibitors. ABCG2 is active as a transporter when bound to the outer membrane of the cell, and in MCF-7 cells, ABCG2 was localised in cell-to-cell junction complexes as well as accumulating in occasional large cytoplasmic vesicles that due to their size and colocalisation with filamentous actin closely resemble aggresomes. DHT treatment of cells reduced ABCG2 mRNA and protein levels and specifically decreased accumulation of ABCG2 in cell-to-cell junction complexes. While ABCG2 mRNA levels were decreased in cyclopamine-treated cells, ABCG2 protein levels remained constant, however ABCG2 localisation in aggresomes was increased, suggesting that ABCG2 function as an efflux transporter was diminished. Efflux of the ABCG2 substrate mitoxantrone was delayed in MCF-7 cells treated with DHT and/or cyclopamine, and the mitoxantrone IC50 was decreased by ~80% in cyclopamine and cyclopamine/DHT treated cells. These findings suggest that novel adjuvant use of androgen and/or Hedgehog signalling inhibitor treatments may augment the activity or duration of treatment responses by inhibiting ABCG2-associated chemoresistance mechanisms.