Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by rapid growth and extensive invasiveness. Overexpression of insulin-like growth factor-binding protein-2 (IGFBP-2) has been reported in GBM. However, it remains to be determined how IGFBP-2 is involved in the progression of GBM. We utilized short hairpin-RNA (shRNA) expression retroviral vectors to inactivate the IGFBP-2 gene permanently in two human GBM cell lines, U251 and YKG-1. The stable knockdown of IGFBP-2 resulted in decreased invasiveness, decreased saturation density of the cells in vitro, and decreased tumorigenicity in nude mice. Transcriptional profiling of both lines revealed several genes that were significantly down-regulated by inactivation of IGFBP-2. One such gene was CD24, which has been implicated in progression of various cancers. Indeed, CD24 was expressed in most GBM cases and the inactivation of CD24 in GBM cells suppressed cellular invasiveness, as was the case for IGFBP-2. Forced overexpression of CD24 led to increased invasiveness of both IGFBP-2-inactivated GBM cell lines and also A172, a human GBM cell line with low endogenous CD24. Further supporting the inter-relationship between IGFBP-2 and CD24, knockdown of IGFBP-2 suppressed the CD24 promoter activity. Moreover, both CD24 promoter activity and in vitro invasiveness were restored in knockdown cells by transfection with an IGFBP-2 expression plasmid. These results indicate that CD24 is modulated by IGFBP-2 and contributes to IGFBP-2-enhanced invasiveness of GBM cells.