The expression of CD44, an adhesion protein associated with the tumor stem cell phenotype, is increased in most human malignant neoplasms. To further delineate the role of CD44 in colon cancer, we inhibited its expression using the siRNA method. HT-29, a human colon cancer cell line producing a large amount of CD44, was transfected with a construct producing a siRNA targeting a 19 mer sequence of the transmembrane domain of CD44 spanning between exon 16 and 17. Following stable transfection, siRNA CD44 resulted in over 75% inhibition of CD44 expression. The stable lines were less adhesive to hyaluronan and more susceptible to apoptosis induced by etoposide. siRNA CD44 clones formed a lower number and size of colonies in soft agar assays. A siRNA CD44 cell clone xenografted in nude mice generated tumors with a reduced tumor volume and wet weight, as compared to control vector clone. Intratumoral gene therapy with a polyethylenimine/siRNA CD44 plasmid DNA complex resulted in tumor growth suppression in nude mice. After siRNA CD44 intratumoral gene therapy, apoptosis was increased in the tumors when compared to the control vector group. In conclusion, based on this mouse xenograft model, siRNA targeting a discrete sequence of human CD44 may provide a potential therapeutic option for colon cancer.