As cancer treatments have shifted toward targeted therapies, the role of developmental signaling pathways in tumorigenesis has garnered special attention. For example, the Hedgehog (Hh) pathway is required for cerebellar development, and its constitutive activation can lead to pediatric medulloblastomas. Hh signaling is normally initiated by the binding of Hh ligands (Shh, Ihh, and Dhh) to the 12TM Patched1 (Ptch1) receptor resulting in accumulation and activation of the G protein-coupled receptor-like protein Smoothened (Smo) within the primary cilium. How Smo regulates Gli transcription factor function remains unclear, but this process involve the scaffolding protein Suppressor of Fused (Sufu), which can directly inhibit the Gli proteins and facilitate their proteolytic processing into N-terminal repressors. Smo is perhaps the most “druggable” target within the Hh pathway, and Smo inhibitors have demonstrated efficacy in murine tumor models and human clinical trials. However, certain cancers can proliferate in response to Gli activation through noncanonical mechanisms and therefore are insensitive to Smo inhibitors. Tumors that initially respond to Smo antagonists can also acquire mutations in Smo or downstream pathway components that render them resistant to these chemotherapies. Chemical inhibitors that act downstream of Smo therefore constitute an important therapeutic strategy for the treatment of Hh pathway-dependent cancers. Most current approaches are biased overwhelmingly to find Smo antagonists or use non-physiological overexpressed Gli cell systems. We overcame these limitations by employing a cell-based reporter that lacks Sufu (Sufu-KO-LIGHT cells) and exhibits constitutive Hh target gene expression in response to endogenous Gli activators to interrogate the MLSMR. We report here for the first time on a novel potent (33.6 nM IC50), non-cytotoxic small molecule antagonist of Gli function that does not inhibit Wnt3a signaling.