The article investigates the modulation of Smoothened (Smo) activity by small molecules, a key protein in Hedgehog (Hh) signaling during embryonic development and tumorigenesis. The authors demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle, antagonizing the inhibitory effects of cyclopamine. Additionally, four structurally distinct small molecules were identified that directly inhibit Smo activity. These compounds exhibit different mechanisms of action, providing insights into the physiological regulation of Smo activity and potential therapeutic applications in Hh signaling disorders. The findings suggest that endogenous small molecules may play a role in regulating Smo function, and the mechanistic differences between the Smo antagonists could be useful for targeted therapeutic interventions.The article investigates the modulation of Smoothened (Smo) activity by small molecules, a key protein in Hedgehog (Hh) signaling during embryonic development and tumorigenesis. The authors demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle, antagonizing the inhibitory effects of cyclopamine. Additionally, four structurally distinct small molecules were identified that directly inhibit Smo activity. These compounds exhibit different mechanisms of action, providing insights into the physiological regulation of Smo activity and potential therapeutic applications in Hh signaling disorders. The findings suggest that endogenous small molecules may play a role in regulating Smo function, and the mechanistic differences between the Smo antagonists could be useful for targeted therapeutic interventions.