The Wnt/β-catenin signaling pathway regulates diverse developmental processes and adult tissue homeostasis. Inappropriate regulation of this pathway has been associated with human diseases, such as cancers, osteoporosis, and Alzheimer`s disease. Using a cell-based chemical screening with natural compounds, we discovered silybin, a plant flavonoid isolated from the Silybum marianum, which activated the Wnt/β-catenin signaling pathway in a synergy with Wnt3a-conditioned medium (Wnt3a-CM). In the presence of Wnt3a-CM, silybin up-regulated β-catenin response transcription (CRT) in HEK293-FL reporter cells and 3T3-L1 preadipocytes through stabilization of intracellular β-catenin protein. Silybin and Wnt3a-CM synergistically reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptorγ (PPARγ) and CAATT enhancer-binding protein α (C/EBPα) in 3T3-L1 preadipocytes, accompanied by the activation of Wnt/β-catenin signaling pathway. Taken together, our findings indicate that silybin is a small-molecule synergist of the Wnt/β-catenin signaling pathway and can be used as a controllable reagent for investigating biological processes that involve the Wnt/β-catenin signaling pathway.