Hyperactivated beta-catenin is a commonly found molecular abnormality in colon cancer, and its nuclear accumulation is thought to promote the expression of genes associated with cellular proliferation and transformation. The p300 transcriptional co-activator binds to beta-catenin and facilitates transcription by recruiting chromatin remodeling complexes and general transcriptional apparatus. We have found that beta-TrCp1/Fbw1a, a member of the Skp1/Cullin/Rbx1/F-box E3 ubiquitin ligase complex, binds directly to p300 and co-localizes with it to beta-catenin target gene promoters. Our data show that Fbw1a, which normally targets beta-catenin for degradation, works together with p300 to enhance the transcriptional activity of beta-catenin, whereas other F-box/WD40 proteins do not. Fbw1a also cooperates with p300 to co-activate transcription by SMAD3, another Fbw1a ubiquitylation target, but not p53 or HIF-1alpha, which are substrates for other ubiquitin ligase complexes. These results suggest that, although Fbw1a is part of a negative feedback loop for controlling beta-catenin levels in normal cells, its overexpression and binding to p300 may contribute to hyperactivated beta-catenin transcriptional activity in colon cancer cells.