In order to study the mechanism of bacteria producing siderophore, B50 was isolated from the cotton rhizosphere. The strain B50 can produce siderophore under iron-limiting conditions. It was identified as Pseudomonas pseudoalcaligenes based on its morphological, physical-chemical characteristics and 16S rDNA sequence. Mutants of B50 were obtained through conjugation. The pyrD gene was cloned from the mutant deficient in siderophores synthesis by TAIL-PCR (Thermal asymmetric interlaced). The pyrD gene is required for the synthesis of dihydroorotate which is a predecessor of deoxyquinoline. Deoxyquinoline was the important part of several siderophores. This is the first report that the Pseudomonas pseudoalcaligenes can produce siderophore.