Rhinoviruses (RV) evoke up to 85% of acute asthma exacerbations in children, 50% in adults, and can induce airway hyperresponsiveness and decrease efficacy of current therapeutics to provide symptom relief. Utilizing human precision cut lung slices (hPCLS), primary human air-liquid interface differentiated airway epithelial cells (HAEC), and human airway smooth muscle (HASM) as pre-clinical experimental models, we demonstrated that RV-C15 attenuates agonist-induced bronchodilation. Specifically, airway relaxation to formoterol and cholera toxin, but not forskolin, was attenuated following hPCLS exposure to RV-C15. In isolated HASM cells, exposure to conditioned media from RV-exposed HAEC decreased cellular relaxation to isoproterenol and PGE2, but not forskolin. Additionally, cAMP generation elicited by formoterol and isoproterenol, but not forskolin, was attenuated following HASM exposure to RV-C15-conditioned HAEC media. HASM exposure to RV-C15-conditioned HAEC media modulated expression of components of relaxation pathways, specifically GNAI1 and GRK2. Strikingly, similar to exposure to intact RV-C15, hPCLS exposed to UV inactivated RV-C15 showed markedly attenuated airways relaxation in response to formoterol, suggesting that the mechanism(s) of RV-C15 mediated loss of bronchodilation is independent of virus replication pathways. Further studies are warranted to identify soluble factor(s) regulating the epithelial-driven smooth muscle loss of beta2-adrenergic receptor (beta2AR) function.