Glioblastoma multiforme (GBM), a lethal brain tumor, contains a small population of GBM stem cells (GSCs), which potentially cause chemotherapeutic resistance and tumor recurrence. Recent studies have reported that incorporation of ribosomes and ribosomal proteins into somatic cells promote lineage trans-differentiation toward multipotency. In this study, we sought to investigate the mechanism underlying stemness acquisition in GBM cells by focusing on 40S ribosomal protein S6 (RPS6). RPS6 was significantly up-regulated in high-grade glioma and localized at perivascular, perinecrotic, and border niches in GBM tissues. siRNA-mediated RPS6 knock-down significantly suppressed the characteristics of GSCs, including their tumor-sphere potential and GSC marker expression. In contrast, RPS6 overexpression enhanced the tumor-sphere potential of GSCs and these effects were attenuated by STAT3 inhibitor (AG490). Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data herein indicated that RPS6 was predominant in GSC niches, concurrent with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were up-regulated in GSC-predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemo-radio-resistance and GBM recurrence.