Objective: Pharmacologic agents designed to promote mucociliary clearance (MCC) in chronic rhinosinusitis (CRS) represent a novel therapeutic strategy. The objectives of the present study were to investigate whether the natural bioflavonoid hesperidin 1) increases transepithelial chloride (Cl) secretion in vitro and in vivo, 2) enhances ciliary beat frequency (CBF), and 3) exerts its mechanistic effects through cAMP/PKA-dependent pathways. Study Design: In vitro and in vivo study. Setting: Laboratory. Subjects and Methods: Transepithelial Cl transport (Ussing chamber) and CBF were investigated in primary murine nasal septal (MNSE) and human sinonasal epithelial (HSNE) cultures. In vivo activity was measured using the murine nasal potential difference (NPD) assay, cystic fibrosis transmembrane conductance regulator (CFTR) R-domain phosphorylation, and cAMP levels were investigated to rule out a cAMP/PKA-dependent mechanism of activation. Results: Hesperidin significantly increased CFTR-mediated Cl transport (change in short-circuit current, ΔISC) in both MNSE (13.51 ± 0.77 vs 4.4 ± 0.66 [control]; P < 0.05) and HSNE (12.28 ± 1.08 vs 0.69 ± 0.32 [control]; P < 0.05). Cl transport across in vivo murine nasal epithelium was also significantly enhanced with hesperidin (2.3 ± 1.0 vs 0.8 ± 0.8 mV [control], P < 0.05). There was no increase in cellular cAMP or phosphorylation of the CFTR R-domain. Hesperidin significantly increased CBF (ratio of pretreatment to post-treatment) with both basal (1.31 ± 0.07 vs 0.93 ± 0.06 [control]; P < 0.05), apical (1.72 ± 0.09 vs 1.40 ± 0.07 [control]; P < 0.05), and basal + apical delivery (2.26 ± 0.18 vs 1.60 ± 0.21, respectively; P < 0.05). Conclusion: Our in vitro and in vivo investigations provide strong support for future testing of this robust Cl secretagogue and CBF activator in human clinical trials for CRS. © 2010 American Academy of Otolaryngology/Head and Neck Surgery Foundation. All rights reserved.