The cell-surface glycoprotein CD44 is closely associated with cell proliferation, tumor invasion, and metastasis. Previous studies have reported that knockdown of CD44 with short hairpin RNA (shRNA) reduced cell proliferation and migration, and induced apoptosis. However, more efficient means of delivering small interference RNA are still necessary. We developed an in vitro model of CD44-shRNA recombinant adenovirus (Ad-CD44-shRNA) and evaluated its ability to alter tumor invasion, migration, and apoptosis in human colon cancer cells. An shRNA against CD44 was used for knockdown of CD44 expression, and recombinant adenovirus was constructed using AD293 cells. The Ad-CD44-shRNA-treated HCT116 colon cancer cells showed a significant decrease in cell proliferation, migration, and invasion, while apoptosis was increased. The Ad-CD44-shRNA also decreased the phosphorylation of Akt and GSK-3β. The levels of Bcl-2 and Bcl-xL expression were downregulated, whereas the expression levels of Bax, cleaved caspase‑3 and -9, and PARP were increased in Ad-CD44-shRNA-treated colon cancer cells. These results support the feasibility of an adenovirus-mediated RNA interference therapy targeting human colon cancer via the CD44 as a potential future therapeutic intervention.