Chemokine (C-C motif) ligand 25 (CCL25) and C-X-C motif chemokine 10 (CXCL10) induce the ligand-specific activation of integrin alpha 4 beta 7 to mediate the selective adhesion of lymphocytes to mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) or vascular cell adhesion molecule-1 (VCAM-1). However, the mechanism underlying the selective binding of different ligands by alpha 4 beta 7 remains obscure. In this study, we demonstrate that CCL25 and CXCL10 induce distinct active conformers of alpha 4 beta 7 with a high affinity for either MAdCAM-1 or VCAM-1. Single-cell force measurements show that CCL25 increases the affinity of alpha 4 beta 7 for MAdCAM-1 but decreases its affinity for VCAM-1, whereas CXCL10 has the opposite effect. Structurally, CCL25 induces a more extended active conformation of alpha 4 beta 7 compared with CXCL10-activated integrin. These two distinct intermediate open alpha 4 beta 7 conformers selectively bind to MAdCAM-1 or VCAM-1 by distinguishing their immunoglobulin domain 2. Notably, Mn2+ fully opens alpha 4 beta 7 with a high affinity for both ligands. Thus, integrin alpha 4 beta 7 adopts different active conformations to switch its ligand-binding specificity.