Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds. At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring opening under mild alkaline conditions.