Abstract Salbutamol is a well-known β2 adrenoceptor (β2AR) partial agonist. We synthesized two boron-containing salbutamol derivatives (BCSDs) with greater potency and efficacy, compared to salbutamol, for inducing β2AR-mediated smooth-muscle relaxation in guinea-pig tracheal rings. However, the mechanism involved in this pharmacological effect remains unclear. In order to gain insight, we carried out binding and functional assays for BCSDs in HEK-293T cells transfected with the human β2AR (hβ2AR). The transfected hβ2AR showed similar affinity for BCSDs and salbutamol, but adenosine 3′,5′-cyclic phosphate (cAMP) accumulation induced by both BCSDs was similar to that elicited by isoproterenol and greater than that induced by salbutamol. The boron-containing precursors (boric and phenylboronic acids, 100 μM) had no significant effect on salbutamol binding or salbutamol-induced cAMP accumulation. These experimental results are in agreement with theoretical docking simulations on lipid bilayer membrane-embedded hβ2AR structures. These receptors showed slightly higher affinity for BCSDs than for salbutamol. An essential change between putative active and inactive conformational states depended on the interaction of the tested ligands with the fifth, sixth and seventh transmembrane domains. Overall, these data suggest that BCSDs induce and stabilize conformational states of the hβ2AR that are highly capable of stimulating cAMP production.