AbstractThe adenosine A₃ receptor (A₃AR), a key member of the G protein-coupled receptor family, is a promising therapeutic target for inflammatory and cancerous conditions. The selective A₃AR agonists, CF101 and CF102, are clinically significant, yet their recognition mechanisms remained elusive. Here we report the cryogenic electron microscopy structures of the full-length human A₃AR bound to CF101 and CF102 with heterotrimeric G_i protein in complex at 3.3-3.2 Å resolution. These agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays reveal the critical role of extracellular loop 3 in A₃AR’s ligand selectivity and receptor activation. Key mutations, including His^3.37, Ser^5.42, and Ser^6.52, in a unique sub-pocket of A₃AR, significantly impact receptor activation. Comparative analysis with the inactive A_2AAR structure highlights a conserved receptor activation mechanism. Our findings provide comprehensive insights into the molecular recognition and signaling of A₃AR, paving the way for designing subtype-selective adenosine receptor ligands.