Mounting evidence supports a major role for the Na/H exchanger NHE1 in cancer progression and metastasis. NHE1 is hyperactive at the onset of oncogenic transformation, resulting in intracellular alkalinization and extracellular microenvironmental acidification. These conditions promote invasion and facilitate metastasis. However, the signal pathways governing the regulation of exchanger activity are still unclear. This is especially important in the aggressively metastatic, triple-negative basal breast cancer subtype. We used affinity chromatography followed by mass spectrometry to identify novel and putative interaction partners of NHE1 in MDA-MB-231 triple-negative breast cancer cells. NHE1 associated with several types of proteins including cytoskeletal proteins and chaperones. We validated protein interactions by co-immunoprecipitation for: 14-3-3, AKT, α-enolase, CHP1, HSP70 and HSP90. Additionally, we used The Cancer Genome Atlas (TCGA) to study NHE1 gene expression in primary patient breast tumours versus adjacent normal tissue. NHE1 expression was elevated in breast tumour samples and, when broken down by breast cancer subtype, NHE1 gene expression was significantly lower in tumours of the basal subtype compared to luminal and HER2+ subtypes. Reverse phase protein array (RPPA) analysis showed that NHE1 expression positively correlated with p90 expression in basal, but not luminal, primary tumours. Other proteins were negatively correlated with NHE1 expression in basal breast cancer tumours. Taken together, our data provides the first insight into the signalling molecules that form the NHE1 interactome in triple-negative breast cancer cells. These results will focus our search for novel targeted therapies.