Abstract Aberrant epidermal growth factor receptor (EGFR) signaling is a typical oncogenic signature in glioblastoma. Here, we show that EGFR inhibition in primary glioma stem cells (GSCs) with oncogenic EGFRvIII and EGFRvIII-transduced glioma stem-like cells promotes invasion by decreasing ID3 levels. ID3 suppresses GSC invasiveness by inhibiting p27KIP1-RhoA-dependent migration and MMP3 expression. Xenograft and human glioblastoma specimens show that ID3 localizes within glioblastoma cores, whereas p27KIP1 and MMP3 are predominantly expressed in glioma cells in invasive fronts. Together, our findings show that EGFR inhibition induces GSC invasiveness by abolishing ID3-mediated inhibition of p27KIP1 and MMP3 expression.