Introduction: Vitamin D is a micronutrient that possesses immunomodulatory actions. Higher vitamin D status has been associated with decreased incidence of acute respiratory infections (ARI) in a number of observational studies. However, mechanistic in vitro work investigating effects of vitamin D on the immune response to ARIs is lacking, especially for rhinovirus, which is the most common respiratory pathogen. Results of clinical trials of vitamin D supplementation in the prevention of ARIs have also been conflicting, in that some demonstrate a protective effect of this intervention against ARI, while others do not. Methods: An immunological assay of ex vivo stimulation with TLR ligands and pathogens in blood samples from participants with asthma, COPD or neither condition in three randomised controlled trials of vitamin D supplementation for the prevention of ARI and exacerbations was developed. This assay was used in conjunction with cellular profiling of clinical trial blood and sputum samples, and a rhinovirus-infected human alveolar cell line (A549 cells) to determine the effects of vitamin D in the protection against acute respiratory infections. Results: The main finding of cell culture experiments was that A549 cells pre-treated with physiological concentrations of 25-hydroxyvitamin D (25[OH]D, the major circulating vitamin D metabolite) had increased resistance to rhinovirus infection, which was associated with attenuation of rhinovirus-induced intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PafR) expression. Immunological analysis of clinical trial samples did not demonstrate any consistent effect of bolus-dose vitamin D supplementation on circulating or pathogen-stimulated inflammatory profiles, or on inflammatory indices in induced sputum. Conclusions: Co-incubation with 25(OH)D was associated with transient protection against rhinovirus infection in a respiratory epithelial cell line in vitro, but these findings did not translate to any changes in cellular profile or inflammatory mediator release in clinical trials samples following in vivo vitamin D supplementation.