Background: Pseudomonas aeruginosa is a gram negative non facultative bacterium and one of the members of normal flora in different sites of body in healthy humans. This bacterium can resist in fluids and hospital environments for a long time.Pseudomonas aeruginosa has two systems for glucose uptake: a low affinity oxidative pathway and a high affinity phosphorylative pathway. The orf BCD genes are located over two million base pair upstream of the genes involved in the high affinity uptake system. Although the role of these genes are unknown by now, they may have a role in regulation of glucose uptake. In the present study, the role of orfD gene in glucose uptake in P.aeruginosa has been investigated.Method: orfD fragment were cloned in pUCP20 as vector and the recombinant plasmid transferred into WMA200 strain of P.aeruginosa, a mutant strain of P.aeruginosa with a chromosomal deletion of orfBCD. So we compared the rate of glucose uptake by P.aeruginosaWMA200, P.aeruginosaWMA200/pUCP20/orfD and P.aeruginosa H103 as wild type strain of P.aeruginosa by using labeled glucose under conditions at low substrate concentration and low cell density.Results: Carbohydrate uptake patterns differed considerably among three strains. The wild type is able to uptake glucose at a faster rate than the mutant; however, the mutant complemented with orfD shows an intermediate uptake comparing to the wild type and the mutant.Conclusion: orfD gene has an important role in carbohydrate uptake in P.aeruginosa strains however further studies are required to determine the involved mechanism.