In humans, deficiency of galactose-1-phosphate uridyltransferase (GALT) can lead a metabolic disorder Classic Galactosemia. Although the biochemical abnormalities associated with this disease have been described in detail, few attempts have been made to characterize the pathogenic mechanisms of this disorder at the molecular level. Here we report the use of high-throughput DNA microarray to examine how galactose affects gene expression in isogenic yeast models that are deficient in either galactokinase (GALK) or GALT, two enzymes which are essential for normal galactose metabolism. We confirmed that the growth of our GALT-deficient, but not GALK-deficient yeast strain ceased 4 h after challenge with 0.2% galactose. Such inhibition was not associated with a reduction of ATP content and was reversible after removal of galactose from medium. We compared the gene expression profiles of the GALT-deficient and GALK-deficient cells in the presence/absence of galactose. We revealed that in the absence of galactose challenge, a subset of genes involved in RNA metabolism was expressed at a level 3-fold lower in the GALT-deficient cells. Upon galactose challenge, significantly more genes involved in various aspects of RNA metabolism and almost all ribosomal protein genes were downregulated in the GALT-deficient, but not GALK-deficient cells. Remarkably, genes involved in inositol biosynthesis and turnover were exclusively induced at high level in the galactose-intoxicated GALT-deficient cells. Our data thus suggested that RNA metabolism, ribosome biogenesis, and inositol metabolism were likely targets for galactose-1-phosphate, a toxic intermediate that is uniquely accumulated under GALT-deficiency.