The histamine H ₄ receptor (H ₄R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H ₃ receptor (H ₃R), two acidic residues in the H ₄R binding pocket, D ^3.32 and E ^5.46, act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H ₄R ligands. Given the symmetric distribution of these complementary pharmacophore features in H ₄R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H ₄R-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 H ₄R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H ₄R 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 H ₄R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H ₄R and can be used as a general approach to elucidate the structure of protein-ligand complexes.