Oestrogen plays a major role in the development and progression of oestrogen receptor-positive breast cancers. Previously, a set of nineteen oestrogen-regulated genes was identified in oestrogen-responsive breast cancer cells. Seventeen were upregulated and two were downregulated. Oestrogen altered the expression of genes that encode proteins involved in the regulation of ion transport. SLC12A2 encodes NKCC1, a Na+-K+-Cl- co-transporter. PDZK1 encodes the scaffolding protein NHERF3 and SCNNIB encodes the β-subunit of the epithelial Na+ channel (ENaC). Expression of the oestrogen-regulated gene SLC12A2 decreased whilst PDZK1 and SCNNIB expression increased. The purpose of this study is to investigate the oestrogen regulation of NKCC1, NHERF3 and ENaC-β protein expression in oestrogen-responsive breast cancer cells by western transfer analysis. NKCC1 expression decreased whereas the expression of NHERF3 and ENaC-β increased in response to oestrogen in all three cell lines MCF-7, EFM-19 and EFF-3. Immunofluorescence data confirmed the localisation of NKCC1 to the cell membrane. NHERF3 and ENaC-β were located mainly in the cytoplasm and there was evidence for vesicular localisation. Functional activity of NKCC1, assessed by 86Rb+ (K+) influx, revealed low activity of NKCC1 which was then enhanced by hypertonicity. NKCC1 activity was decreased more by oestrogen treatment in EFM-19 cells than in MCF-7. Intracellular pH was measured with a pH-sensitive dye BCECF-AM to evaluate Na+/H+ exchange. Oestrogen increased the capacity of EFM-19 cells to regulate pHi. The oestrogenic and anti-oestrogenic effects of several anti-oestrogens were assessed. Notably, bazedoxifene was agonistic for NKCC1 expression. Tamoxifen, 4-hydroxytamoxifen, toremifene and lasofoxifene were partially agonistic for NHERF3. The antagonistic activities of the anti-oestrogens were more complex and varied between cell lines for all three proteins. Fulvestrant was unable to reverse the oestrogen-induced expression of ENaC-β. In conclusion, this study confirmed that oestrogen alters the expression and activity of proteins involved in ion transport. Modification of this cell phenotype may favour breast cancer progression.