We previously showed that pharmacological activation of adenosine monophosphate activated protein kinase (AMPK) inhibited Na+ transport in H441 human airway epithelial cells. However, the effect of physiological activators has not been investigated. Removal of glucose from the medium (glucose deprivation; < 0.4 mM) for 4 hours increased phospho/total acetyl CoA carboxylase (ACC) (a target for AMPK mediated phosphorylation) as analysed by western blotting from 0.5 ± 0.06 to 0.9 ± 0.13 densitometry units, P = 0.04, n = 4 in H441 cells. In addition, phospho/total AMPK was correspondingly increased. Glucose deprivation significantly decreased spontaneous short circuit current across H441 monolayers to 80.8 ± 5.30 % (P = 0.008, n = 8 of the controls), without any significant effect on the resistance of the monolayers. Glucose deprivation also significantly reduced amiloride-sensitive transepithelial Na+ transport from 29.1 ± 2.56 to 21.6 ± 0.19 µA/cm2, P < 0.009, n = 8. In the presence of Compound C (80 μM), a pharmacological inhibitor of AMPK, amiloride-sensitive transepithelial Na+ transport was reduced from 23.2 ± 2.67 to 6.9 ± 0.75 µA/cm2 by glucose deprivation. It was significantly lower than monolayers deprived of glucose alone (P < 0.009, n = 8). Interestingly, Western blotting demonstrated that Compound C further increased phospho/total ACC induced by glucose deprivation significantly from 1.3 ± 0.12 to 2.7 ± 0.23 densitometry units, P = 0.001, n = 4. Phospho/total AMPK was also correspondingly increased. These data indicate that glucose deprivation increased AMPK activity and this correlated with decreased ion transport in H441 cell monolayers. However, that Compound C augmented the effect of glucose deprivation indicates that care must be taken when interpreting the results obtained from its use. Supported by the BBSRC and St George’s University of London.