Emerging evidence indicates that the Warburg effect (aerobic glycolysis) is a marker of malignancy in pancreatic cancer (PC). Receptor expression-enhancing protein 3 (REEP3) is dysregulated in various cancers; however, no studies have addressed whether REEP3 is involved in regulating PC malignancy, particularly with respect to the Warburg effect. Herein, we identified a new diagnostic marker of PC, which may be useful for developing drugs to treat PC and providing insight into the molecular pathology of PC. The microarray dataset GSE183795 was retrieved from the Gene Expression Omnibus database and the differentially expressed genes were analyzed. REEP3 expression was examined in PC cell lines and normal pancreatic ductal epithelial cells. Following REEP3 knockdown or overexpression in PC cells, cell invasion, migration, glucose uptake, lactate production, ATP levels, and epidermal growth factor receptor (EGFR)/extracellular regulated kinase (ERK) pathway protein expression were assessed via scratch, Transwell, glucose, and lactate assays and western blot analysis. A database analysis revealed that REEP3 was highly expressed in PC tumor tissues from 179 patients. In addition, patients with high REEP3 tumor expression exhibited poor overall and disease-free survival. Our results suggest that REEP3 is upregulated in multiple PC cell lines. Moreover, cell viability indicators, such as proliferation, invasion, migration, glycolytic activity, and EGFR/ERK pathway protein expression were conspicuously restrained on REEP3 disruption in PC cells. Overall, these findings suggest that REEP3 assists PC cell proliferation, invasion, and migration by facilitating the Warburg effect through the activation of the EGFR/ERK signaling pathway.