Heptosyltransferase I (HepI) is a glycosyltransferase bacterial protein part of the GT-B class of enzymes. It is responsible for the transfer of a heptose moiety from ADP-L-glycero-D-manno-heptose (ADPH) to Kdo2-Lipid A as part of the inner oligosaccharide core biosynthesis of lipopolysaccharides. HepI has been broadly characterized through work done in Taylor lab which laid the groundwork for the biophysical studies done. The primary goal of this thesis is to use these biophysical studies to aid in the discovery and characterization of inhibitors of HepI. A clinically relevant inhibitor for HepI has been sought for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. Inhibition of LPS biosynthesis would yield bacteria with a cellular membrane exhibiting higher permeability for hydrophobic antibiotics that can enhance the efficacy for treating resistant species of bacteria. While many labs have discovered or designed novel small molecule inhibitors for glycosyltransferases, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein through CD thermal melt analysis, small-angle x-ray scattering studies, and site-directed mutagenesis has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A through molecular dynamics simulation studies suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes and protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. These studies, in addition to in vivo validation of aminoglycoside activity, suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI which is evidenced by observations of modification of LPS production in vivo. Further research explored ADPH analogs, with both α- and β- configurations of their glycosidic linkage that could potentially act as ligands of HepI. While some acted as inhibitors of HepI like the second generation α-analog, others acted like good substrates for HepI instead. Finally, studies involving an alternative GT-B enzyme, MshA the D-inositol 3-phosphate glycosyltransferase which has similar structural and dynamic characteristics to HepI, was studied to expand our understandings of HepI to other GT-B enzymes as well. It is evident, that studies done here on HepI were successful in further understanding the dynamics of the enzyme to inhibit it. Biologically relevant nanomolar inhibitors of HepI were revealed and characterized both in vitro and in vivo which led to the discovery of a novel mechanism of action for aminoglycoside antibiotics. In addition, second generation ADPH analogs were examined and shown to inhibit HepI with the potential to be potent transition-state analog inhibitors of HepI. These findings will ultimately impact the scientific community focused on antimicrobial design and discovery as HepI remains a potent target to increase cell membrane permeability and aminoglycoside inherent mechanism of action targets HepI and the LPS pathway.