We have previously shown that the enhancement of glucose uptake caused by the inhibition of gap junctional communication is a consequence of the increase in astrocyte proliferation. Since C6 glioma cells are highly proliferative and are poorly coupled through gap junctions, we used these cells to investigate the effect of increasing gap junctional communication on the rate of glucose uptake. Previous work by us had shown that tolbutamide increases gap junctional communication in C6 glioma cells, as does dbcAMP, a classical activator of gap junctional communication. In this work, our results show that both tolbutamide and dbcAMP reduce the rate of glucose uptake in C6 glioma cells and that their effects are additive. The main glucose transporters expressed in C6 glioma cells are GLUT-1 and GLUT-3. Neither the expression nor the cellular localization of either GLUT-1 or GLUT-3 were modified by increasing gap junctional communication. The estimation of glucose uptake with 2-deoxyglucose includes not only glucose transport but also glucose phosphorylation, which in C6 glioma cells is mainly catalyzed by type I and type II hexokinase. Our results reveal that the increase in gap junctional communication caused by tolbutamide and dbcAMP is associated with a decrease in the activity of hexokinase. In agreement with this, tolbutamide and dbcAMP caused a rapid change in the localization of both type I and type II hexokinase, which were detached from the mitochondria to the cytosol.