Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor and has been described for G protein-coupled receptors. However, it has not yet been described for ligands interacting with integrins without αI domain. Here, we show by molecular dynamics simulations that four side chain-modified derivatives of tauroursodeoxycholic acid (TUDC), an agonist of α₅β₁ integrin, differentially shift the conformational equilibrium of α₅β₁ integrin towards the active state, in line with the extent of β₁ integrin activation from immunostaining. Unlike TUDC, 24-nor-ursodeoxycholic acid (norUDCA)-induced β₁ integrin activation triggered only transient activation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase and, consequently, only transient insertion of the bile acid transporter Bsep into the canalicular membrane, and did not involve activation of epidermal growth factor receptor. These results provide evidence that TUDC and norUDCA exert a functional selectivity at α₅β₁ integrin and may provide a rationale for differential therapeutic use of UDCA and norUDCA.