AbstractAim: Caffeine and theophylline inhibit phosphatidylinositol 3‐kinase (PI3‐kinase) activity and insulin‐stimulated protein kinase B (PKB) phosphorylation. Insulin‐stimulated glucose uptake involves PI3‐kinase/PKB, and the aim of the present study was to test the hypothesis that caffeine and theophylline inhibit insulin‐stimulated glucose uptake in skeletal muscles.Methods: Rat epitrochlearis muscles and soleus strips were incubated with insulin and different concentrations of caffeine and theophylline for measurement of glucose uptake, force development and PKB phosphorylation. The effect of caffeine was also investigated in muscles stimulated electrically.Results: Caffeine and theophylline completely blocked insulin‐stimulated glucose uptake in both soleus and epitrochlearis muscles at 10 mm. Furthermore, insulin‐stimulated PKB Ser473and Thr308and GSK‐3β Ser9phosphorylation were blocked by caffeine and theophylline. Caffeine reduced and theophylline blocked insulin‐stimulated glycogen synthase activation. Caffeine stimulates Ca2+release and force development increased rapidly to 10–20% of maximal tetanic contraction. Dantrolene (25 μm), a well‐known inhibitor of Ca2+‐release, prevented caffeine‐induced force development, but caffeine inhibited insulin‐stimulated glucose uptake in the presence of dantrolene. Contraction, like insulin, stimulates glucose uptake via translocation of glucose transporter‐4 (GLUT4). Caffeine and theophylline reduced contraction‐stimulated glucose uptake by about 50%, whereas contraction‐stimulated glycogen breakdown was normal.Conclusion: Caffeine and theophylline block insulin‐stimulated glucose uptake independently of Ca2+release, and the likely mechanism is via blockade of insulin‐stimulated PI3‐kinase/PKB activation. Caffeine and theophylline also reduced contraction‐stimulated glucose uptake, which occurs independently of PI3‐kinase/PKB, and we hypothesize that caffeine and theophylline also inhibit glucose uptake in skeletal muscles via an additional and hitherto unknown molecule involved in GLUT4 translocation.