Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion. Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking, suggesting compensation by other NHE family members. Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis.