Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. However, numerous diseases have been attributed to the aberrant transduction of Wnt signaling. We screened a plant extract library targeting TCF/β-catenin transcriptional modulating activity with a cell-based luciferase assay. Activity-guided fractionation of the MeOH extract of the E. longifolia root led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 exhibited TCF/β-catenin inhibitory activity. Compound 1 decreased the expression of Wnt signal target genes, mitf and zic2a, in zebrafish embryos. Treatment of SW480 cells with 1 decreased β-catenin and increased phosphorylated β-catenin (Ser 33, 37, Tyr 41) protein levels. The degradation of β-catenin by 1 was suppressed by GSK3β-siRNA, while compound 1 decreased β-catenin even in the presence of CK1α-siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3β independent of CK1α.