Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis and an inducer of neural differentiation. We previously reported the loss of PEDF expression in glioma progression. In this study, we investigated whether PEDF overexpression could suppress glioma growth and invasion. Glioma cell line U251 was stably transfected with a full-length human PEDF expression vector. The expression and release of various cytokines and angiogenic factors into the medium were analyzed by real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. Apoptosis was checked by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Growth inhibition was evaluated by using the in vitro Matrigel invasion. Tumorigenicity was examined in vivo by subcutaneous xenotransplantation into severe combined immunodeficient mice. In U251 cells overexpressing PEDF, thrombospondin-1 protein was upregulated (5.3-fold more), but the production of vascular endothelial growth factor (VEGF) (1.8-fold less) and basic fibroblast growth factor (2.5-fold less) was lower than in cells transfected with the vector only. PEDF also downregulated the production of matrix metalloproteinase-9. Conditioned medium collected from the PEDF-transfected U251 cells showed a significant reduction of VEGF expression. In vitro invasiveness was reduced by approximately 40%. PEDF expression prevented the growth of transfected cells and caused a significant increase in the percentage of cells undergoing apoptosis (50.4% in PEDF-transfected cells). Furthermore, the size of xenotransplants was significantly smaller. In conclusion, PEDF overexpression decreased malignancy, and this might be attributed to the promotion of apoptosis and the regulation of expression of angiogenic effectors. Thus, treatment with PEDF may be useful in patients with malignant gliomas. However, the mechanism of apoptosis induction needs to be investigated.