Glabrescione B is a novel compound that specifically binds to Gli1, an important modulator of the Hedgehog (Hh) signaling pathway. By disrupting the Gli1-DNA interaction, Glabrescione B effectively impairs the activity of Gli1. Moreover, Glabrescione B demonstrates potent inhibitory effects on the growth of tumor cells that rely on Hedgehog signaling, as well as on the self-renewal capacity and clonogenicity of tumor-derived stem cells.