Glucose concentration in the airway surface liquid (ASL) is normally lower than blood (~0.4mM compared to ~5mM). We developed an in vitro model of airway glucose homeostasis and showed that, normally, glucose diffuses from blood/interstitial fluid across the respiratory epithelium into the ASL via paracellular pathways, and this is limited by epithelial permeability, glucose uptake and metabolism. In this model, increasing the diffusion gradient for glucose across the epithelium (e.g. hyperglycaemia) elevated glucose in ASL [1, 2]. Sugars (glucose and fructose) are known to promote the growth of S. aureus [3]. The growth of S. aureus on the apical surface of an in vitro model of human airway epithelium (H441) was significantly increased by elevating basolateral glucose concentration from 10mM to 20mM or 40 mM (colony forming units; cfu increased by 155±49% or 392±105%, p<0.05 and p<0.001, respectively, n=9). Furthermore, there were significantly more bacteria post infection in the airways of an in vivo mouse model of diabetes (db/db) (4611±995 bronchoalveolar lavage; BAL cfu) than wild type mice (950±293 BAL cfu, p<0.001, n=9). To confirm that S. aureus utilised sugars that diffused across the epithelium into ASL, we obtained several S. aureus JE2 derived strains from the Nebraska Transporon Mutant Library which contained genetically disrupted genes associated with sugar transport. We firstly characterised the effect of these mutations on growth in the presence of sugars. Addition of either fructose (10 mM) or glucose (10 mM) significantly increased the growth of the parent strain (JE2) (measured as log10 OD600) at 8 hours (p<0.001, n=3, respectively). Glucose (10 mM) promoted the growth of all S. aureus strains, although two strains, NE1944 and NE172, with mutations in the glucose specific domain of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), exhibited mildly reduced growth at 8 hours (p<0.05, n=3). NE768 a strain in which the fructose specific permease had been disrupted, exhibited significant growth inhibition in the presence of fructose compared to glucose (p<0.001, n=3) consistent with an inability to utilise fructose to support its growth. Comparison of sugar induced JE2 and NE678 growth in bacterial epithelial co-culture showed that basolateral fructose increased apical JE2 but not NE678 growth at 7 hours (p<0.05, n=5). These data provide new evidence that fructose in addition to glucose diffuses across the airway epithelium and is utilised by S. aureus as a substrate for growth. This provides a proof of concept for S. aureus utilisation of glucose in ASL. Our data also indicate that pathways for glucose uptake in S. aureus are more complex and may involve multiple mechanisms.