Human GK(beta) (pancreatic beta-cell glucokinase) is the main glucose-phosphorylating enzyme in pancreatic beta-cells. It shares several structural, catalytic and regulatory properties with Hxk2 (hexokinase 2) from Saccharomyces cerevisiae. In fact, it has been previously described that expression of GK(beta) in yeast could replace Hxk2 in the glucose signalling pathway of S. cerevisiae. In the present study we report that GK(beta) exerts its regulatory role by association with the yeast transcriptional repressor Mig1 (multicopy inhibitor of GAL gene expression 1); the presence of Mig1 allows GK(beta) to bind to the SUC2 (sucrose fermentation 2) promoter, helping in this way in the maintenance of the repression of the SUC2 gene under high-glucose conditions. Since a similar mechanism has been described for the yeast Hxk2, the findings of the present study suggest that the function of the regulatory domain present in these two proteins has been conserved throughout evolution. In addition, we report that GK(beta) is enriched in the yeast nucleus of high-glucose growing cells, whereas it shows a mitochondrial localization upon removal of the sugar. However, GK(beta) does not exit the nucleus in the absence of Mig1, suggesting that Mig1 regulates the nuclear exit of GK(beta) under low-glucose conditions. We also report that binding of GK(beta) to Mig1 allows the latter protein to be located at the mitochondrial network under low-glucose conditions.