Recent studies have demonstrated that gastric cancer stem cells (CSCs) are a rare sub-group of gastric cancer (GC) cells and have an important role in promoting the tumor growth and progression of GC. In the present study, we demonstrated that the glycolytic enzyme Enolase 1 (ENO1) was involved in the regulation of the stem cell-like characteristics of GC cells, as compared to the parental cell lines PAMC-82 and SNU16, the expression of ENO1 in spheroids markedly increased. We then observed that ENO1 could enhance stem cell-like characteristics, including self-renewal capacity, cell invasion and migration, chemoresistance, and even the tumorigenicity of GC cells. ENO1 is known as an enzyme that is involved in glycolysis, but our results showed that ENO1 could markedly promote the glycolytic activity of cells. Furthermore, inhibiting glycolysis activity using 2-deoxy-D-glucose treatment significantly reduced the stemness of GC cells. Therefore, ENO1 could improve the stemness of CSCs by enhancing the cells' glycolysis. Subsequently, to further confirm our results, we found that the inhibition of ENO1 using AP-III-a4 (ENOblock) could reduce the stemness of GC cells to a similar extent as the knockdown of ENO1 by shRNA. Finally, increased expression of ENO1 was related to poor prognosis in GC patients. Taken together, our results demonstrated that ENO1 is a significant biomarker associated with the stemness of GC cells.