The Wnt signaling pathway plays diverse roles in embryonic development and adult homeostasis, whereas aberrant activation of Wnt signaling may lead to tumorigenesis. In this research, we explored for natural products that inhibit Wnt activity. Activity-guided fractionation of MeOH extract of C. gigantea exudates yielded six cardenolides including calotropin. These cardenolides exhibited inhibitory activities TCF/β-catenin transcriptional activity with IC50 0.7 to 3.8 nM, and also showed cytotoxicity against three colorectal cancer cell lines (DLD1, HCT116, and SW480). Treatment of SW480 colon cancer cells with calotropin led to a significant decrease of β-catenin protein in a dose-dependent manner. However, the addition of MG-132, a proteasome inhibitor, abolished the effect of calotropin. It was revealed that calotropin induced increase of not only mRNA expression but also protein level of Casein Kinase 1α (CK1α). Calotropin also induced phosphorylation of β-catenin on CK1α site (S45) and GSK-3β site (S33, S37, T41). However, treatment with CKI-7 (CK1α inhibitor) and CK1α RNAi obstructed degradation of β-catenin by inhibition of CK1α and decreasing of CK1α protein level, respectively. These results suggested that calotropin inhibited Wnt signal activity through increase of protein level of CK1α.