Na+/H+ exchanger isoform 1 (NHE1) is known to play a key role in regulating intracellular pH and osmotic homeostasis and is involved in the development and progression of several types of cancer. However, the function and specific mechanism of NHE1 in gastric cancer (GC) are not clearly understood. In the present study, we report that NHE1 is overexpressed in tissues and cell lines from GC patients, and knockdown or inhibition of NHE1 suppressed GC cell proliferation via regulation of G1/S and G2/M cell cycle phase transitions, concomitant with a marked decrease in positive cell cycle regulators, including cyclin D1 and cyclin B1. Likewise, NHE1 was required for GC cell migration and invasion through the regulation of epithelial-mesenchymal transition (EMT) proteins, and NHE1 inhibition resulted in an acidic intracellular environment, providing possible mechanisms underlying NHE1-mediated GC progression both in vitro and in vivo. These data highlight the important role of NHE1 in GC progression and suggest that NHE1 may be a useful target for GC therapy.