Enolase-phosphatase 1 (ENOPH1), is an enzyme that is involved in polyamine biosynthesis and is associated with stress responses. However, little is known about its role in the pathophysiology of glioma. In the present study, we examined the expression and function of ENOPH1 in human glioma tissues and cell lines. Western blot, qPCR and immunohistochemistry analysis were performed to investigate the expression of the ENOPH1 protein in glioma tissues in 86 patients. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), wound healing and cell cycle assays were implemented to identify cell growth and cell migration in U87 and U251 glioma cells. The results revealed that compared with normal brain tissues, the level of ENOPH1 was markedly increased in glioma tissues. In addition, we observed that the glioma pathological grade was positively associated with the expression level of ENOPH1. Knockdown of ENOPH1 expression with siRNA markedly reduced cell proliferation, and significantly decreased cell migration. Notably, knockdown of ENOPH1 promoted its downstream protein, aci-reductone dioxygenase 1 (ADI1), to shift from the nucleus to the cytoplasm of U251 glioma cells, while MT1-MMP expression was significantly downregulated compared with the control group. Collectively, our data demonstrated that the knockdown of ENOPH1 suppressed cell growth and migration, which may be associated with ADI1 translocation and MT1-MMP downregulation in glioma cells. Thus, ENOPH1 could serve as an underlying therapeutic target of glioma.