The A2A adenosine receptor (A2AAR) is a unique G-protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A2AAR-antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S672.65, M1775.38, and N2536.55, while KW6002 binds to F622.60, A813.29, and H2647.29. Moreover, interactions with L1675.28 are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo-A2AARs were found to be different from the apo-A2AAR in three typical functional switches, (i) the “ionic lock” was in equilibrium between formation and breakage in apo-A2AAR, but stayed broken in holo-A2AARs; (ii) the “rotamer toggle switch,” T883.36/F2426.44/W2466.48, adopted different rotameric conformations in apo-A2AAR and holo-A2AARs; (iii) apo-A2AAR preferred α-helical intracellular loop (IC)2 and flexible IC3, while holo-A2AARs had a flexible IC2 and α-helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo-A2AAR and holo-A2AARs. Proteins 2013; 81:1399–1410. © 2013 Wiley Periodicals, Inc.