Phosphatidylethanolamine (PE)‐binding protein 4 (PEBP4) is an antiapoptotic protein that is aberrantly expressed in various malignancies. We previously demonstrated that PEBP4 expression is dramatically induced in human gliomas and positively correlated with tumor grade and patient survival. However, the function of PEBP4 in human glioma development and underlying mechanisms remain largely unknown. By stable lentiviral vector‐mediated silencing of PEBP4, we examined the effects of PEBP4 knockdown on the growth, apoptosis, and invasion of U251 and U373 human glioma cell lines using MTT, Transwell, colony formation, and flow cytometric assays. We examined the in vivo role of PEBP4 in tumor growth by inoculation of BALB/c nu/nu male mice with PEBP4‐deficient U251 and U373 cells. The expression of cell cycle‐ and apoptosis‐related proteins was analyzed by Western blotting and immunostaining. Knockdown of PEBP4 significantly reduced the proliferation and invasion of human glioma cells while inducing cell apoptosis by altering the expression of cell cycle‐ and apoptosis‐related proteins. Mechanistically, PEBP4 knockdown led to activation of the extracellular signal‐regulated kinases 1/2 (ERK1/2) pathway, an effect that could be reversed by U0126, a selective inhibitor of MEK1/2 (upstream of ERK1/2), suggesting involvement of ERK1/2 signaling in the regulation of glioma development and progression by PEBP4. We identified PEBP4 as a novel regulator mediating human glioma cell proliferation, invasion, and apoptosis as well as tumor formation and growth. Therefore, PEBP4 may be a potential therapeutic target in human glioma treatment.