The study identifies two novel classes of small molecules that disrupt Wnt pathway responses: one inhibits Porcupine (Porcn), a membrane-bound acyltransferase essential for Wnt protein production, and the other abrogates Axin protein destruction, a suppressor of the Wnt/β-catenin pathway. These compounds enable a chemical genetic approach to study Wnt pathway responses and stem cell function in adult tissue. The IWP compounds block Wnt secretion, while the IWR compounds affect β-catenin levels. Biochemical studies show that IWP compounds target Porcn, which is required for Wnt protein maturation, and IWR compounds stabilize Axin proteins, promoting β-catenin destruction. In vivo experiments demonstrate that IWR compounds can transiently and reversibly suppress Wnt/β-catenin pathway responses in zebrafish, affecting tissue regeneration and stem cell maintenance. Additionally, IWR compounds inhibit aberrant Wnt/β-catenin activity in cancer cells, potentially through stabilization of Axin proteins. These findings highlight the chemically tractable regulatory mechanisms within the Wnt pathway and suggest their potential for therapeutic applications.The study identifies two novel classes of small molecules that disrupt Wnt pathway responses: one inhibits Porcupine (Porcn), a membrane-bound acyltransferase essential for Wnt protein production, and the other abrogates Axin protein destruction, a suppressor of the Wnt/β-catenin pathway. These compounds enable a chemical genetic approach to study Wnt pathway responses and stem cell function in adult tissue. The IWP compounds block Wnt secretion, while the IWR compounds affect β-catenin levels. Biochemical studies show that IWP compounds target Porcn, which is required for Wnt protein maturation, and IWR compounds stabilize Axin proteins, promoting β-catenin destruction. In vivo experiments demonstrate that IWR compounds can transiently and reversibly suppress Wnt/β-catenin pathway responses in zebrafish, affecting tissue regeneration and stem cell maintenance. Additionally, IWR compounds inhibit aberrant Wnt/β-catenin activity in cancer cells, potentially through stabilization of Axin proteins. These findings highlight the chemically tractable regulatory mechanisms within the Wnt pathway and suggest their potential for therapeutic applications.