The cancer stem cell (CSC) model suggests that a small subset of cancer cells possess stem cell properties and plays a crucial role in tumor initiation, metastasis and resistance to anticancer therapy. Exploration of the specific therapies targeting at CSCs has been a crucial issue in antitumor research. Gastric cancer (GC) cells often exist in an ischemic microenvironment with acidic conditions in vivo, thus maintenance of cellular pH homeostasis is important for the survival and function of GC cells. Proton pump inhibitors (PPIs) may prevent intracellular proton extrusions which consequently reduce cancer cell survival under acidic conditions. The effects of PPIs on the suppression of the viability and invasiveness of GC cells have been reported, but the functional role of pantoprazole (PPZ) in GC cells remains unknown. In this study, we found that when cells were treated with PPZ, the 5‑fluorouracil (5‑FU) chemosensitivity was upregulated, meanwhile the sphere formation ability and the relative expression levels of stem cell markers CD44, CD24, ABCG2, EpCAM and Lgr5 were significantly decreased. It was hypothesized that PPZ inhibits the GC CSCs. Successively a sphere formation culture was performed to establish CSC models and the effect of PPZ on GC CSCs from SGC-7901 and HGC‑27 cells was explored. The addition of PPZ reduced the relative expression of CSC markers and anti‑drug markers accompanied by a decrease in proliferation, 5‑FU chemoresistance and self‑renewal capacity via epithelial‑mesenchymal transition (EMT)/β‑catenin pathways. The study suggests that PPZ could be a promising novel specific therapeutic strategy for targeting GC CSCs.