Introduction Gastric cancer (GC) was recorded as the third leading cause of cancer-related deaths worldwide, and overall and recurrent survival after potentially curative gastrectomy for advanced gastric cancer remains poor. Thus, developing the GC targeting molecules in combination with adjuvant radiotherapy, or adjuvant chemotherapy following surgical resection is an important trend for GC treatment. Material and methods We compared isobaric tags for relative and absolute quantification (iTRAQ) proteomics data from 6 GC samples with those of 86 GC samples from the Chen dataset and consequently identified 122 intersecting/overlapping proteins that were highly expressed in gastric cancer tissue from both datasets followed by Kaplan-Meier survival analysis. To explore the downstream effectors of GEF-X, we analysed thousands of genes in relation to GEF-X expression from three GC datasets published in the literature. Genes displaying positive correlation with GEF-X were subsequently analysed using Metacore analysis. Results and discussions Clinical findings indicated that higher GEF-X expression is positively correlated with tumour size, depth of invasion, lymph node metastasis, vascular invasion and pathological stage. Elevated GEF-X expression was associated with significantly shorter cumulative survival of two independent cohorts of GC patients in both univariate and multivariate analyses. Depletion of GEF-X suppressed cell migration, proliferation, sphere formation, anchorage-independent growth, radioresistance and tumorigenicity as well as expression of gastric cancer stem cell (CSC) markers. GEF-X depletion was accompanied by a concomitant decrease in nuclear b-catenin and CD44 levels. Moreover, interactions between GEF-X and β-catenin were observed in the nucleus of GC cells. Conclusions Our results suggest for the first time a novel role of GEF-X in the self-renewal of CSCs, supporting its utility as an independent marker of GC progression.GEF-X depletion was accompanied by a concomitant decrease in nuclear b-catenin and CD44 levels. Moreover, interactions between GEF-X and β-catenin were observed in the nucleus of GC cells.