Constitutive activation of the Wnt/β-catenin pathway is thought to play a central role in colorectal carcinogenesis. A key output in this pathway is the nuclear level of β-catenin, which determines the transcription of T-cell transcription factor (TCF)/lymphoid enhancer-binding factor-responsive target genes. In unstimulated cells, β-catenin is continuously targeted for ubiquitin-dependent degradation, which depends on its NH₂-terminal phosphorylation by glycogen synthase kinase-3β (GSK-3β) in association with a multiprotein complex. Previously, we have shown that the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin and indomethacin down-regulate β-catenin/TCF signaling in colorectal cancer cells. Here, we demonstrate that the reduced signaling activity of β-catenin in response to NSAIDs is a result of its enhanced phosphorylation. In SW948 and SW480 colorectal cancer cells, phosphorylation of NH₂-terminal S/T residues time dependently increased in response to aspirin and indomethacin. In contrast, in 293 cells, NSAID treatment failed to induce detectable levels of β-catenin phosphorylation but resulted in degradation of β-catenin within 24 h in serum-deprived cells. The aspirin-induced β-catenin phosphorylation in colon cancer cells preceded down-regulation of β-catenin/TCF signaling, suggesting a causal relationship. Inhibition of this process by LiCl pointed to participation of GSK-3β. Unexpectedly, GSK-3β was also phosphorylated upon aspirin treatment in six colorectal cancer cell lines. We present evidence that inactivation of a phosphatase rather than stimulation of a kinase or interference with the ubiquitination machinery may be the cause of the stabilized phosphorylation. The data emphasize the importance of β-catenin in the pathogenesis of colorectal cancer and define it as a key target for anticancer therapeutics.