Bitter taste receptor‐14 (TAS2R14) is a GPCR also expressed on human airway smooth muscle cells, which signals to intracellular [Ca 2+ ], resulting in relaxation of the airway, and is a novel target for bronchodilators. Here, we examine long‐term, agonist‐promoted down‐regulation of TAS2R14 expression because tachyphylaxis would be an undesirable therapeutic characteristic. Five TAS2R structurally distinct full agonists were studied to ascertain biasing away from down‐regulation. Agonist exposure for 18 h caused minimal desensitization by diphenhydramine (DPD) compared with ~50% desensitization with all other agonists. Agonists evoked β‐arrestin recruitment to TAS2R14, which was not seen with a phosphoacceptor‐deficient mutant, TAS2R14‐10A. All agonists except for DPD also caused subsequent TAS2R14 internalization and trafficking via early and late endosomes to down‐regulation. TAS2R14‐10A failed to undergo these events with any agonist. Molecular docking showed that DPD has specific interactions deep within a binding pocket that are not observed with the other agonists, which may lock the receptor in a conformation that does not internalize and therefore does not undergo down‐regulation. Thus, TAS2R14 is subject to β‐arrestin‐mediated internalization and subsequent down‐regulation with chronic exposure to most agonists. However, by manipulating the agonist structure, biasing toward G‐protein coupling but away from long‐term down‐regulation can be achieved.—Woo, J. A., Castaño, M., Goss, A., Kim, D., Lewandowski, E. M., Chen, Y., Liggett, S. B. Differential long‐term regulation of TAS2R14 by structurally distinct agonists. FASEB J. 33, 12213‐12225 (2019). www.fasebj.org