Glucose is the preferred carbon source for cells, and the molecular machinery responsible for glucose uptake and metabolism is well understood. In contrast, how cells reprogram genome-wide expression when they encounter glucose is not known. We used genome-wide expression analysis to elucidate the global regulatory response to glucose and to investigate how negative regulators participate in glucose repression. Our results reveal that 6% of yeast genes are induced and 8% are repressed by exposure to glucose. We find that the negative regulatory response is largely effected through the combined action of two negative regulators that act on TATA-binding protein (NC2 and Mot1), components of the RNA polymerase II holoenzyme (Srb10 and Sin4), a factor recruited to promoters by sequence-specific DNA-binding proteins (Tup1) and histones. Although previous studies have established that proper expression of many genes depends on the combined action of multiple positive regulators, these results provide evidence for combinatorial regulation of a substantial portion of the genome through highly conserved negative regulators.