UDP-galactose 4'-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia.