We conducted a study on molecular epidemiology and clinical implications of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolated from urine. Over a 10-year period from 2001 through 2010, a total of 92 MBL-producing P. aeruginosa urine isolates were collected from patients (one isolate per patient) who were admitted to 5 hospitals in Okayama Prefecture, Japan. When cross-infection was suspected in the hospital, pulsed-field gel electrophoresis was performed. In the resulting dendrogram of 79 MBL-producing P. aeruginosa urine isolates, no identical isolates and 7 pairs of isolates with >80% similarity were found. The biofilm-forming capabilities of 92 MBL-producing P. aeruginosa urine isolates were significantly greater than those of 92 non-MBL-producing urine isolates in a medium of modified artificial urine. The imipenem resistance transferred in 16 of 18 isolates tested, and these frequencies were in the range of 10⁻³ to 10⁻⁹. All of 18 isolates tested belonged to internationally spread sequence type 235 and had 3 gene cassettes of antimicrobial resistance genes in the class 1 integron. The strong biofilm-forming capabilities of MBL-producing P. aeruginosa urine isolates could be seriously implicated in nosocomial infections. To prevent spread of the organism and transferable genes, effective strategies to inhibit biofilm formation in medical settings are needed.