Key residues and binding mechanisms of PGE and PGE on prostanoid receptors are poorly understood due to the lack of X-ray structures for the receptors. We constructed a human EP3 (hEP3) model through integrative homology modeling using the X-ray structure of the β-adrenergic receptor transmembrane domain and NMR structures of the thromboxane A2 receptor extracellular loops. PGE and PGE docking into the hEP3 model showed differing configurations within the extracellular ligand recognition site. While PGE could form possible binding contact with S211, PGE is unable to form similar contacts. Therefore, S211 could be the critical residue for PGE recognition, but is not a significant for PGE. This prediction was confirmed using HEK293 cells transfected with hEP3 S211L cDNA. The S211L cells lost PGE binding and signaling. Interestingly, the S211L cells retained PGE-mediated signaling. It indicates that S211 within the second extracellular loop is a key residue involved in turning down PGE signaling. Our study provided information that S211L within EP3 is the key residue to distinguish PGE and PGE binding to mediate diverse biological functions at the initial recognition step. The S211L mutant could be used as a model for studying the binding mechanism and signaling pathway specifically mediated by PGE.