The human histamine H₃ receptor (hH₃R) is predominantly expressed in the CNS, where it regulates the synthesis and release of histamine and other neurotransmitters. Due to its neuromodulatory role, the hH₃R has been associated with various CNS disorders, including Alzheimer’s and Parkinson’s disease. Markedly, the hH₃R gene undergoes extensive splicing, resulting in 20 isoforms, of which 7TM isoforms exhibit variations in the intracellular loop 3 (IL3) and/or C-terminal tail. Particularly, hH₃R isoforms that display variations in IL3 (e.g., hH₃R-365) are shown to differentially signal via Gα_i-dependent pathways upon binding of biased agonists (e.g., immepip, proxifan, imetit). Nevertheless, the mechanisms underlying biased agonism at hH₃R isoforms remain unknown. Using a structure-function relationship study with a broad range of H₃R agonists, we thereby explored determinants underlying isoform bias at hH₃R isoforms that exhibit variations in IL3 (i.e., hH₃R-445, -415, -365, and -329) in a Gα_i-dependent pathway (cAMP inhibition). Hence, we systematically characterized hH₃R isoforms on isoform bias by comparing various ligand properties (i.e., structural and molecular) to the degree of isoform bias. Importantly, our study provides novel insights into the structural and molecular basis of receptor isoform bias, highlighting the importance to study GPCRs with multiple isoforms to better tailor drugs.