Na+/H+ exchanger 1 (NHE1; SLC9A1) is a transport protein responsible for pH regulation in various types of cells. In cardiac myocytes, NHE1 generates the largest transmembrane acid-extrusion flux and is therefore the most significant controller of the intracellular acid/base milieu. The protein itself is regulated by various enzymes, including kinases. A number of critically important sites have been identified, coupling NHE1 to signalling cascades such as cAMP. NHE1 activity is very sensitive to oxygen tension, thus linking metabolism with pH. However, the sites implicated in this effect are not known. This study identified novel phosphorylation sites on NHE1. The state of these residues regulates NHE1 activity, hence pH and a myriad of pH sensitive downstream processes. Dysregulated NHE1 activity has been shown in diseases of the heart. Identification of novel regulatory sites can help demarcate the mechanisms of disease and suggest possible interventions to correct NHE1 activity.