Glucose in the airway surface liquid (ASL) is normally maintained at a low concentration compared to the blood (~12.5 x lower). Raised blood glucose elevates ASL glucose (1), which increases the risk of respiratory infection, particularly with methicillin-resistant Staphylococcus aureus (2). We investigated the relationship between basolateral/blood glucose concentration and apical/luminal S. aureus growth using an in vitro model of human airway epithelium and an in vivo mouse model and tested whether metformin (drug used to lower blood glucose) affected this relationship. H441 epithelial cells grown at air-liquid-interface were inoculated with 5×105 CFU/cm2 S. aureus (8325-4) on the apical surface and incubated for 7 hours. The transepithelial electrical resistance (TEER) of H441 monolayers was significantly reduced and basolateral to apical paracellular glucose flux across H441-monolayers (assessed using 14C-L-glucose) was greatly enhanced following co-incubation with S. aureus (P<0.05 and P<0.0001 respectively, n=8). A 12h pre-treatment of H441 cells with metformin (1mM) partially attenuated the fall in TEER and decreased transepithelial glucose flux (P<0.001 and P<0.0001 respectively, n=8). Apical S. aureus growth (determined by Miles-Misra) increased with basolateral glucose concentration (10, 20, 40mM). However, no correlation was seen in metformin treated co-cultures, and S. aureus growth was significantly impaired (P<0.05, n=8). Metformin treatment had no effect on bilateral 14C-D-glucose uptake in H441 monolayers or any direct effect on S. aureus growth in culture. 6-10 week old wild type C57BL/6 (WT) or db/db (leptin receptor deficient) mice were treated with intraperitoneal PBS or 40mg/kg metformin for two days prior to intranasal inoculation with 107CFU S. aureus. Mice were culled 24h post infection and bronchoalveolar lavage (BAL) collected. The db/db mice had significantly higher blood glucose concentrations than WT mice (P<0.01, n=9). Metformin had no effect on blood glucose levels in either the WT or db/db mice. Untreated db/db mice had more S. aureus in their BAL than WT mice (P<0.001, n=9). Metformin treatment significantly reduced numbers of S. aureus in the BAL of db/db mice compared to PBS treated db/db mice (P<0.01, n=9). Metformin had no effect on S. aureus CFU in the BAL of WT mice. These results indicate that metformin treatment inhibits S. aureus growth in the airways by reducing the paracellular diffusion of glucose into the ASL under hyperglycaemic conditions.