Besides being a pH_i regulator, Na⁺/H⁺ exchanger 1 (NHE1) has recently been postulated as a membrane scaffold that assembles protein complexes and coordinates various signaling pathways. The aim of the current study is to uncover NHE1 interactive partners and study their functional implications. NHE1 interactive partners were screened in the mouse brain using a signal transduction antibodyarray. Ten out of 400 tested proteins appeared to be potentially associated with NHE1. These partners have been shown to be involved in either cell proliferative or apoptotic pathways. The interactions between NHE1 and src homology 2 domain-containing tyrosine phosphatase (SHP-2), Bin 1 and heat shock protein 70 (HSP70) were reciprocally confirmed by co-immunoprecipitation. Moreover, in vitro binding data have shown that NHE1 C-terminus interacts directly with SHP-2. The functional significance of the association between NHE1 and SHP-2 was further investigated by measuring intracellular pH (pH_i), cell proliferation, and cell death using the fluorescent dye BCECF, [³H]-thymidine incorporation, and medium lactate dehydrogenase activity, respectively. Our results revealed that cells with SHP-2 over-expression exhibited a higher steady-state pH_i and a faster, NHE1-dependent pH_i recovery rate from acid load in HEPES buffer. In addition, SHP-2 over-expression diminished the HOE-642-induced inhibition of cell proliferation and protected cells from hypoxic injury, especially in the presence of HOE-642. Taken together, our findings demonstrate that SHP-2 is not only physically associated with NHE1 but also modulates NHE1 functions such as pH_i regulation, cell proliferation and cell death under hypoxia.