Human genome-wide association studies (GWASs) have identified over 50 loci associated with pulmonary function and related phenotypes, yet follow-up studies to determine causal genes or variants are rare. Single nucleotide polymorphisms (SNPs) in serotonin receptor 4 (HTR4) are associated with human pulmonary function in genome-wide association studies and follow-up animal work has demonstrated that Htr4 is causally associated with pulmonary function in mice, although the precise mechanisms were not identified. We sought to elucidate the role of neural innervation and pulmonary architecture in the lung phenotype of Htr4^-/- animals. We report here that the Htr4^-/- phenotype in mouse is dependent on vagal innervation to the lung. Both, ex vivo tracheal ring reactivity and in vivo flexiVent™ pulmonary functional analyses demonstrate that vagotomy abrogates the Htr4^-/- airway hyperresponsiveness phenotype. Hyperpolarized ³He gas magnetic resonance imaging and stereological assessment of wild-type and Htr4^-/- mice reveal no observable differences in lung volume, inflation characteristics, or pulmonary-microarchitecture. Finally, control of breathing experiments reveal substantive differences in baseline breathing characteristics between mice with/without functional HTR4 in breathing frequency, relaxation time, flow rate, minute volume, time of inspiration and expiration and breathing pauses. These results suggest that HTR4's role in pulmonary function likely relates to neural innervation and control of breathing.