Smoothened (Smo), a G protein-coupled receptor family member, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Cyclopamine, a plant-derived steroidal alkaloid, directly inhibits Smo, suggesting endogenous small molecules may regulate Smo. This study demonstrates that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle and antagonizes cyclopamine. Four small molecules, structurally distinct from cyclopamine, directly inhibit Smo activity. These compounds provide insights into Smo regulation and may have therapeutic potential for Hh signaling modulation. Hh signaling specifies embryonic pattern by directing cellular differentiation and proliferation, while aberrant activation is linked to tumors like basal cell carcinoma and medulloblastoma. Smo and Patched (Ptc) are integral membrane proteins that mediate Hh responses. Hh binds to Ptc, relieving its suppression of Smo, which then activates downstream events, including the stabilization of Cubitus interruptus (Ci) and expression of Ci-dependent genes. Smo is a single vertebrate homologue involved in all Hh signaling. Despite Smo's central role, mechanisms regulating its activation remain unclear. Studies show that Hh stimulation alters Smo phosphorylation and subcellular localization, but the relationship to activation is not known. Ptc inhibits Smo function, possibly catalytically. Structural perturbations in oncogenic Smo mutants (e.g., W539L) cause constitutive pathway activation. Recent studies suggest Smo regulation may involve endogenous small molecules. Cyclopamine binds to Smo's heptahelical domain and antagonizes Hh signaling. Ptc is structurally related to resistance-nodulation-cell division (RND) family proteins and Niemann-Pick C1 (NPC1), which transport hydrophobic compounds, suggesting Ptc may control Smo function by influencing its interactions with small molecules. To study Smo activation, the study identified and characterized small molecules modulating Smo function. SAG, a chlorobenzothiophene molecule, binds to the Smo heptahelical bundle and acts downstream of Ptch1 in the Hh pathway. SAG counteracts cyclopamine inhibition of Smo and activates the Hh pathway. SAG binds directly to Smo, as shown by photoaffinity labeling and fluorescence binding assays. SAG's binding site is localized to the Smo heptahelical bundle, and it binds with comparable affinity to Smo, Smo ΔCRD, and Smo ΔCT. Four small molecules (SANT-1 to SANT-4) were identified as Smo antagonists. These compounds inhibit Smo activity by binding to the heptahelSmoothened (Smo), a G protein-coupled receptor family member, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Cyclopamine, a plant-derived steroidal alkaloid, directly inhibits Smo, suggesting endogenous small molecules may regulate Smo. This study demonstrates that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle and antagonizes cyclopamine. Four small molecules, structurally distinct from cyclopamine, directly inhibit Smo activity. These compounds provide insights into Smo regulation and may have therapeutic potential for Hh signaling modulation. Hh signaling specifies embryonic pattern by directing cellular differentiation and proliferation, while aberrant activation is linked to tumors like basal cell carcinoma and medulloblastoma. Smo and Patched (Ptc) are integral membrane proteins that mediate Hh responses. Hh binds to Ptc, relieving its suppression of Smo, which then activates downstream events, including the stabilization of Cubitus interruptus (Ci) and expression of Ci-dependent genes. Smo is a single vertebrate homologue involved in all Hh signaling. Despite Smo's central role, mechanisms regulating its activation remain unclear. Studies show that Hh stimulation alters Smo phosphorylation and subcellular localization, but the relationship to activation is not known. Ptc inhibits Smo function, possibly catalytically. Structural perturbations in oncogenic Smo mutants (e.g., W539L) cause constitutive pathway activation. Recent studies suggest Smo regulation may involve endogenous small molecules. Cyclopamine binds to Smo's heptahelical domain and antagonizes Hh signaling. Ptc is structurally related to resistance-nodulation-cell division (RND) family proteins and Niemann-Pick C1 (NPC1), which transport hydrophobic compounds, suggesting Ptc may control Smo function by influencing its interactions with small molecules. To study Smo activation, the study identified and characterized small molecules modulating Smo function. SAG, a chlorobenzothiophene molecule, binds to the Smo heptahelical bundle and acts downstream of Ptch1 in the Hh pathway. SAG counteracts cyclopamine inhibition of Smo and activates the Hh pathway. SAG binds directly to Smo, as shown by photoaffinity labeling and fluorescence binding assays. SAG's binding site is localized to the Smo heptahelical bundle, and it binds with comparable affinity to Smo, Smo ΔCRD, and Smo ΔCT. Four small molecules (SANT-1 to SANT-4) were identified as Smo antagonists. These compounds inhibit Smo activity by binding to the heptahel