The essential role of pathogens in host metabolism is widely recognized, yet the mechanisms by which they affect host physiology remain to be fully defined. Here, we found that NIeB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to possess N-acetylglucosamine (GlcNAc) transferase activity, GlcNAcylates HIF-la, a master regulator of cellular O-2 homeostasis. We determined that NIeB-mediated GlcNAcylation at a conserved arginine 18 (Arg18) at the N-terminus of HIF-1 alpha enhanced HIF-1 alpha transcriptional activity, thereby inducing HIF-1 alpha downstream gene expression to alter host glucose metabolism. The arginine transferase activity of NIeB was required for its enhancement of HIF-1 alpha transactivity and the subsequent effect on glucose metabolism in a mouse model of EPEC infection. In addition, HIF-1 alpha acted as a mediator to transact NIeB-mediated induction of glucose metabolism-associated gene expression under hypoxia. Thus, our results further show a causal link between pathogen infection and host glucose metabolism, and we propose a new mechanism by which this occurs.