The histamine H₄ receptor (H₄R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H₃ receptor (H₃R), two acidic residues in the H₄R binding pocket, D^3.32 and E^5.46, act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H₄R ligands. Given the symmetric distribution of these complementary pharmacophore features in H₄R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H₄R–ligand binding modes by combining (3D) quantitative structure–activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4­(5)-imidazolyl)­propyl]­isothiourea) derivatives to investigate H₄R–ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H₄R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H₄R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H₄R and can be used as a general approach to elucidate the structure of protein–ligand complexes.