Iron-limiting conditions have been reported to be prevalent in the milieu of urinary tract. In the present investigation, effect of iron on virulence of uropathogenic Pseudomonas aeruginosa in planktonic and biofilm cell mode was studied. Significant enhancement in elaboration of all the virulence traits along with increased adherence to uroepithelial cells and decreased phagocytosis of P. aeruginosa was observed following growth in iron-deplete medium. On the contrary, decrease in all these parameters except phagocytosis was observed when P. aeruginosa was grown in iron-rich medium. In vivo, P. aeruginosa grown in iron-deplete medium showed increased renal bacterial load and tissue pathology in a mouse model of ascending urinary tract infection compared with organisms grown in iron-replete medium. The results of the present study may help in understanding host-parasite interaction and in developing alternative preventive approach against P. aeruginosa induced urinary tract infections.