Abnormal regulation of Wnt/beta-catenin signaling followed by increased levels of the beta-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit beta-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the beta-catenin mRNA. We designed a strategy to identify the cleavage sites in beta-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzyme-working sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave beta-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of beta-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited beta-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to beta-catenin significantly reduced beta-catenin-dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against beta-catenin might have potential as a therapeutic intervention to treat colon cancer.