The authors investigated the anticancer activity of penfluridol in glioma sphere-forming cell lines (GSCs) in vitro, a subpopulation of GBM cells responsible for treatment resistance and GBM recurrence, and reported an inhibitory effect of penfluridol on GLI-1, which subsequently reduced the GLI-1-associated markers of stemness (SOX2, NESTIN, OCT4), markers of invasion (Integrin α6, uPAR), and epithelial-mesenchymal transition (EMT) factors (Vimentin, Zeb-1, N-cadherin, Snail, Slug), molecules strongly associated with increased cell motility and resistance to genotoxic agents of GSCs. This evidence concerns the gene PLAUR and glioblastoma.