The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and through phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the relative positioning of the two GFAT-1 domains to control its activity. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.