Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. Smo^D473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with Smo^D473H, and the molecular docking results showed that B13 could bind Smo^D473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by Smo^D473H mutations.