Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosa contains d-rhamnosylated O-antigen. The synthesis of GDP-D-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-D-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-D-mannose, and then reduced to GDP-D-rhamnose by GDP-4-keto-6-deoxy-D-mannose reductase (RMD). Here, we describe the enzymatic characterization of P. aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S. cerevisiae allowed us to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. Thus, coexpression of the Helicobacter pylori gmd and P. aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose. This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy. The functional expression of P. aeruginosa RMD in S. cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.