The current work handles the comparative characterisation of five distinct organ barriers in regard to transport and inhibition of glucose. The organs of the lung, placenta, liver and intestines were represented by cancer cell lines in in-vitro barriers models. As the transport of glucose is controlled by various active and passive glucose transporters, different inhibitors of the passive facilitate glucose transporter (GLUT) family and/or the active sodium-glucose linked transporter (SGLT) family were tested. Prior, the barrier integrity was assessed with the transepithelial/endothelial resistance and sodium fluorescein leakage assays. Cytochalasin B (CB) reduced sugar transport by 52% in the placental BeWo b30 and by 42% in the hepatic Hep G2 barrier due to inhibition of the highly expressed passive transporters GLUT1 and GLUT3. A moderate inhibition of 34% of GLUT1, 3 4 and 12 in the lung A549 barrier with CB was identified. The intestinal barriers Caco-2 and HT-29 showed an overall low inhibition of 19% with CB as GLUT2 and SGLT1 transporters were not inhibited and presumably responsible for the majority of glucose transport apart from GLUT1. In contrast, phloretin (PT) binds to the receptors of GLUT2 and SGLT1 and achieved a reduction of up to 45% in the intestinal cancer barriers. However, due to its lower inhibitory strength in GLUT1 and GLUT4, overall lower inhibition rates of 20% in lung, 38% in placental and 36% in hepatic barriers were accomplished. The results suggest that in placental and hepatic barriers the glucose transfer is mainly contributed to GLUT1 and GLUT3, and the glucose transport in intestinal barriers to GLUT2 and SGLT1.