Wnt signaling is essential for embryogenesis and adult tissue homeostasis and contributes to cancer development, especially in the colon. A new essential pathway component, legless, was identified in Drosophila melanogaster. Mice with Cre/loxP-mediated conditional ablation of the legless homologues Bcl9 and Bcl9l in the intestinal epithelium were generated to assess the contribution of the genes to Wnt signaling and their potential suitability as therapeutic drug targets. Initial analysis had revealed that both genes were not required for self-renewal of the intestinal epithelium during homeostasis, but for regeneration of experimentally induced colon wounds. Furthermore, formation of chemically induced colon tumors was not impaired and Bcl9/Bcl9-mutant tumors were morphologically indistinguishable from wild-type tumors and displayed hallmarks of active Wnt signaling. This study aimed at further investigating the molecular processes involving Bcl9 and Bcl9l in wound regeneration and tumorigenesis in the colon, and in particular relatedness of the genes to Wnt signaling. To this end, transcriptomes of laser microdissected wound regions were analyzed by microarray-based gene expression profiling and realtime quantitative PCR, but did not reveal significant changes in gene expression at the time point chosen for analysis. However, expression profiling of homeostatic colon epithelium revealed a reduced expression of several intestinal stem cell marker genes in Bcl9/Bcl9l-mutant colon epithelium, suggesting that the regeneration deficit has been caused by functional impairment of intestinal stem cells. This stem cell deficiency did not differ between young and old mice and was also weakly detectable in small intestinal epithelium. To verify that tumors originated from cells in which complete deletion of all four Bcl9 and Bcl9l alleles had occurred and to assess Wnt signaling activity more comprehensively, laser microdissected tumor regions were profiled using microarrays and realtime quantitative PCR. This analysis confirmed that Bcl9 and Bcl9l were not required for colon tumorigenesis and revealed that despite the morphological similarity, transcriptional profiles of Bcl9/Bcl9l-mutant tumors were very different from wild-type tumors. Notably, loss of Bcl9 and Bcl9l caused reduced expression of a subset of direct Wnt target genes, genes related to epithelial-mesenchymal transition (EMT) and intestinal stem cell traits. While wild-type tumor epithelium appeared to be in a metastable state, characterized by co-expression of epithelial and mesenchymal traits, Bcl9/Bcl9l-mutant tumor epithelium predominantly expressed epithelial traits. In addition, a marked reduction in expression of several genes associated with malignant tumor properties was observed in Bcl9/Bcl9l-mutant tumors. This was highlighted by the preservation of an intact basement membrane around Bcl9/Bcl9l-mutant tumor epithelium, which was absent in wild-type tumors, and indicated that loss of Bcl9/Bcl9l decreased tumor aggressiveness. Of note, EMT and stemness traits are correlated to tumor resistance to therapy and thus interfering with Bcl9/Bcl9l function might restore drug responsiveness. As a proof of concept, it was shown that the malignant traits mediated by Bcl9/Bcl9l could be abrogated in wild-type tumors by inducing deletion of the genes. These findings suggest that targeting Bcl9 and Bcl9l with therapeutic drugs has the potential to attenuate the malignancy of colon tumors.