At various stages during embryogenesis and cancer cells are exposed to tension, compression and shear stress; forces that can regulate cell proliferation and differentiation. In the present study, we show that shear stress blocks cell cycle progression in colon cancer cells and regulates the expression of genes linked to the Wnt/β-catenin, mitogen-activated protein kinase (MAPK) and NFκB pathways. The shear stress-induced increase of the secreted Wnt inhibitor DKK1 requires p38 and activation of NFκB requires IκB kinase-β. Activation of β-catenin, important in Wnt signaling and the cause of most colon cancers, is inhibited by shear stress through a pathway involving laminin-5, α6β4 integrin, phosphoinositide 3-kinase (PI 3-kinase) and Rac1 coupled with changes in the distribution of dephosphorylated β-catenin. These data show that colon cancer cells respond to fluid shear stress by activation of specific signal transduction pathways and genetic regulatory circuits to affect cell proliferation, and indicate that the response of colon cancers to mechanical forces such as fluid shear stress should be taken into account in the management of the disease.